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OI'SOXINS    AND   \'A('('IXI-:s    IX    MKDICIXI-:   AXD   Sl'I.MlKin'. 


\(»i;.\IAN    KDW  .\i;i)    DITMAX,   A.M..    M.D.. 

IN    (OM.Ar.oii.vrio.v    with 

XATll.WlKl.    I'..    l-OTTKi;.    .M.J). 

ERXKST  ]!.  l'.i;.\l)LKV.  M.D. 


Suliiiiitlcd  ill  I'ailial  KuHilluiciil  of  tlic  i;c(|uirciiu'iils  l(ir  tlic  Dcuifc  <'l  Dottur 
lit   Pliiliisdphy  ill  tlic  Ka<'iilty  of  Piiic  Science.  C'nhinihia   rnivcrsity. 

Ill  ]8!K")  Deny!?  and  Leclef^  iirst  called  attention  to  the  efPect  of  blood 
serum  on  phagocytosis  by  knicocytes.  From  tlie  careful  experiments  ("/( 
rilro  ihev  concluded  that  in  an  immunized  ralihit  the  leucocytes  obtain 
I  heir  power  of  engulfing  the  bacteria  from  some  property  of  the  serum. 
The  immunized  animal  fights  tlie  bacteria,  first,  l)y  the  direct  action  of 
its  serum;  second,  by  its  leucocytes.-  The  latter  always  owe  to  the 
serum  the  commencement  of  their  power. 

In  189T  Bordet-'  was  nnable  to  confii'in  their  results.  In  th.e  same 
year  ^lennes^  showed  that  the  immunity  of  guinea-pigs  inoculated  with 
toxins  or  cultures  of  jjneumococci  depends  on  a  modiiication  of  their 
serum  whereby  an  active  phagocytosis  is   induced,   and   that  this   is  not 


1.  "Sur  le  mecanisme  tie  I'iuimuiiite  chez  le  lapin  vaccine  coutre  le  snv;il()ci)ccii>: 
pyog.,"  La  Cellule,  1895,  vol.  xl,  p.  198. 

2.  A   summary   of  their  conclusions   is  as  follows  : 

(ai  The  serum  of  normal  ralibits  exercises  no  bactericidal  action  on  tlic  siiri>tit- 
cvcciis   iiyogenvs. 

(b)  The  .serum,  of  a  rabbit  on  whom  a  certain  degree  of  immunity  to  strepto 
coccus  has  been  conferred  by  vaccination,  will  delay  the  development  of  streptococcus 
for   several   hours   and  will   sometimes   exhibit   a   true  bactericidal   power. 

(c)  Leucocytes  from  a  nornuil  rabbit  added  to  (he  serum  of  a  normal  rabbii 
exhibit  only  a  feeble  phagocytic  power  to  streptococcus.  The  leucocyles  die  lieforc 
their  usual    term   of  life. 

(dj  Leucocytes  from  a  normal  ral)l)it  added  to  the  serum  of  a  vaccinated  rabbii 
cuergetically  destroy  streptococcus.  Tlie  leucocytes  preserve  their  normal  duration 
of  life. 

(e)  Leucocytes  from  a  vaccinated  ratil)it  added  to  its  own  serum  act  the  same 
as   above    (d). 

(f)- Leucocytes  from  a  vaccinated  ra'oltit  added  to  the  serum  of  a  normal  rabl)it 
act   as  above    (c). 

(g)  If  a  dose  of  streptococcus,  let^hal  for  a  normal  rabbit,  is  injected  into  the 
pleura   of  a  vaccinated  ralibit.   the   serum   prevents   the  development   of  a   pleurisy. 

(h)  If  a  dose  of  streptoioccus  capable  of  producing  erysipelas  in  a  normal  rabbit 
is  injected  under  the  skin  of  a  vaccinated  ralibit  the  infection  is  prevented  especially 
by    leucocytes. 

3.  Ann.    de   I'lnstitat    I'asteiir.    ISIIT.   p.    liol.    note. 

4.  "Antipneumokokken  Serum  und  der  Mechanismus  der  Ininuinitiit  des  Kanincbcn 
gegen    den    Tneumokokkus."    ysclu'.    f.    Ilyg..    1897.    toI.    xxv.    j).    41:;. 


183478 


r^ 


o^ 


due  to  any  special  activity  of  ilic  leucocytes.  Ja  100'^  Leishnian''  devised 
a  luetliod  of  ([Uantitativeiy  estimating  the  j)lia<iocytic  ])o\ver  of  Ijlood  to 
staphylococci.  In  190;5  \\'ri<ilit  aiul  Douglas"  introduced  the  word 
"opsonin"  {upsono,  I  jjrepare  the  food  for)  to  charjicterize  the  substance 
in  normal  blood  Avhicli  they  believed  prejjared  the  microljes  for  ingestion 
by  tlie  phagocytes.  'J'hey  were  able  to  sliow  that  this  substance  exists  iii 
the  serum,  that  it  is  partly  therniolabile,  being  largely  destroyed  at  60° 
('.  in  ten  minutes,  and  that  it  acts  on  the  bacteria  ami  not  on  the  leuco- 
cytes.' 

In  1JK»4  Neufeld  and  iJinipau.'^  in<le|ien(lently  of  W'riglit.  alluded  to 
the  two  well-known  elements  in  immune  sera  (i.  e..  antitoxin  and  bac- 
tericidal substances)  and  stated  that  tlu'y  had  foiuid  a  third  I'lenient 
which  they  claimed  sensitized  the  bacteria,  but  did  not  act  on  the  leuco- 
cytes. The}'  treated  leucocytes  with  antistreptococcus  seruiu  for  a  certain 
time,  suspended  them  in  normal  serum,  and  found  that  they  did  not 
ingest  virulent  streptococci  (note  that  they  employed  virulent  organ- 
isms). On  the  other  hand,  after  treating  virulent  streptococci  Avith  anti- 
streptococcus  serum  and  then  washing  them  free  from  the  serum  with 
salt  solution,  they  fonnd  lencocytes  could  then  actively  ingest  these  sensi- 
tized microbes.  They  obtained  corresponding  results  with  pneunu)cocci. 
They  proposed  to  call  their  sensitizing  substances  "bacteriotropic**  sub- 
stances. To  these  sid^stances  AVright  and  Douglas  have  given  the  name 
"immune  opsonins."' 

Miri' irons    of    i:sTi:MATi.\(i    iMiAoorvrosis    qrA\TiTATi\i;i.v. 

In  \iH)-i  Leislunan"'  first  devised  a  method  for  (piantitat  i\cly  eslimat- 
ing  phagocytosis.  Tie  mixed  e<pial  quantities  of  blood  and  a  suspension 
of  staphylococci  in  salt  solution  in  ca])illary  ])ipettes,  l)le\v  out  a  drop  of 
the  mixture  on  the  end  of  a  glass  slide,  on  tlie  other  end  a  similar  mix- 
tui'e.  using  (-out rol  blood,  covered  with  covei'  glasses  and  iiicuhalt'd  for 
oiu'-half  hour  in  a  moist  chamber.     After  slidimz'  off  the  co\er  i^lasscs  the 


.">.   "Motlind   of   lOslimatins   I'liagocytip    Powit,"    nri(.    Med.    .loin-..    T.kpl',    p,    7.'!. 
t>.   I'roc.    of   IJoyal    Soc,    vol.    l.xxii,   p.   ^o~ . 

7.   Tl»<'    foUowinfr   pxperimfiils    were    rpsponsihic   for    llich-   conclusions  : 
(ii)    Staph.vlfn'occiis   •'Uiulsioii     •     washed    corpuscles    +    .\a("l    solulioii    ai    :!7    C.    I'd- 
1.")  minutes  =  no  phagocylosis.      Add  scrum;  phagocytosis  occurs. 

(b)  'riu-pp  vols,  washed  coi-pii.scles  +  :>  vols,  serum  al  :!7  t '.  fni-  1.",  uiiiuilcs.  ilicu 
heated  to  (>()  (".  (dest.-oyiiiR  the  thermolahiie  opsonin  i,  Cool  |  1  vi.l.  emulsion 
staphylococci   at  ;'.7   (".   for    1."»   minnlcs.   result    is   little   or   no   phajiocylosis. 

(c)  Three  vols,  serum  -|-  1  vol.  emulsion  slajihylococci  at  :>7  (".  for  1 .1  minutes, 
then  heated  to  »;()  C.  +  a  vols,  (orpuscles  at  :'.7  (".  for  1.'.  minutes,  result  is  marked 
phaKocytosis.  IIenct>.  action  of  serum  is  mainly  to  modify  the  bacteria  in  such  a 
way   as   to  render   them   a   pr<-y   to   the   iihauocyiosis. 

The  organisms  which  are  ilius  acted  on  are  staphylococci,  si  rcptoco.-ri.  yonococcus 
pneumococciis,  MU-iiivih-ciih  tiicUttimis.  vibrio  of  Asiatic  cholera,  and  colon,  pest! 
typhoid,   tubercle,   anthrax,   lu-oteu.s   and  dysentery   bacilli. 

M.  Xeitfeld  and  Itimpau  ;  "fel)er  die  Antil<orper  der  S(reptol<ol<U,.u  und  I'neumo- 
kokken  Iinmunseruui,"  Deutsch.  med,  Wochschr..  4(1,  1it(»4.  Sept  !".•  p  1  t.'.S  •  also 
/tschr.   f.    llvj:..    I'.Mi.-..   vol    ii,   p    -js:',. 


preparation  was  lixed  and  stained.     By  counting  tlic  cocci  in  a  certain 
number  of  leucocytes  an  average  per  leucocyte  was  obtained,  which  com- 
pared with  the  control  constituted  the  "phagocytic  index." 
Wright's*'  modification^  of  this  method  is  as  follows : 

Equal  volumes  of  (1)  a  bacterial  emulsion  in  salt  solution;  (2)  washed 
blood  corpuscles;  and  (3)  the  serum  to  be  tested  are  measured  in  a  capillary 
pipette  fitted  with  a  rubber  teat,  mixed  thoroughly  by  blowing  out  on  a  slide 
several  times,  drawn  up  in  the  same  pipette,  the  end  sealed  in  the  flame,  and 
incubated  for  fifteen  or  twenty  minutes  at  37  C.  A  drop  of  the  mixture  is  then 
blown  out  on  a  slide,  and  a  smear  fixed  and  stained.  The  enumeration  of  the 
bacteria  in  twenty  to  fifty  polymorphonuclear  neutrophiles  is  made  under  an  oil 
immersion  lens  and  an  average  per  leucocyte  obtained.  The  ratio  of  this  average 
to  that  for  a  normal  serum,  using  the  same  emulsion  of  leucocytes  and  corpuscles, 
constitutes  the  "opsonic  index.'"" 

In  general  tlie  procedure  for  making  the  bacterial  suspension  is  as 
follows  :^i 

TECHXIQCE. 

In  the  ease  of  staphylococci,  streptococci,  jjneumococci,  gonococci,  and  colon 
bacilli,  the  twenty-four  hour  growth  of  agar  slants  is  washed  off  with  normal 
salt  solution.*  This  is  allowed  to  settle  and  the  upper  part  of  the  turbid  fluid 
is  pipetted  off  and  centrifugalized  in  a  small  tube  for  from  five  to  eight  minutes 
at  high  speed.  The  slightly  opalescent  supernatant  fluid  is  then  carefully  taken 
off  in  a  pipette  and  is  used  for  the  bacterial  suspension. 

In  the  case  of  tubercle  bacilli,  it  is  necessary  to  proceed  a  little  differently. 
The  conglomerated  mass  of  bacilli  is  heated  at  100  C.  for  ten  minutest  and  a 
loopful  is  ground  up  in  an  agate  mortar,  adding  from  time  to  time  a  drop  of 
salt  solution  until  about  2  c.c.  have  been  added.  This  is  centrifugated  as  in 
the  ease  of  the  cocci  for  from  five  to  eight  minutes,  until  the  supernatant  fluid 
is  only  slightly  opalescent.  This  is  pipetted  off  and  used  for  the  bacterial  sus- 
pension. 

The  washed  corpuscles  are  obtained  by  receiving  thirty  to  forty  drops  of 
blood^-  into  about  30  c.c.  of  a  1  per  cent,  sodium  citrate  in  0.85  per  cent,  salt 
solution.  This  prevents  coagulation.  The  corpuscles  are  swung  completely  down 
in  the  centrifuge  and  are  washed  twice  in  0.85  per  cent,  salt  solution  to  remove 
all  traces  of  serum  and  citrate.  After  the  final  washing,  all  the  salt  solution 
is  pipetted  off  and  the  upper  layer  of  corpuscles,  which  is  rich  in  leucocytes,  is 
taken  for  the  "washed  corpuscles." 

The  serum  is  obtained  by  receiving  the  blood  in  small  glass  capsules  with 
curved  capillary  limb,  allowing  it  to  clot  and  then  centrifugating  so  that  the 
clear  serum  can  be  easily  removed. 


it.  '-Blood  Fluids  in  Coiinection  with  riiagocytosis,"  Ibid.,  Sept,  1,  1003,  vol. 
ixxiii.  p.  130. 

10  Simon  (Johns  Hopkins  Hospital  Reports,  January.  1906)  has  still  further  mod- 
ified this  technic  by  making  successive  dilutions  of  the  control  and  test  sera  and  com- 
paring the  points  at  which  phagocytosis  is  practically  abolished,  or  by  comparing 
the  percentages  of  leucocytes   engaged  in  phagocytosis   in  a  given   dilution. 

11.  The  details  of  our  technic  are  essentially  ihose  of  Wright,  to  whom  we  wish 
to  express  our  thanks  for  his  kindness  in  personally  explaining  his  methods  to  one 
of  us. 

♦Wright  informs  us  that  he  now  employs  much  younger  cultures  of  the  strep- 
tococci, gonococci  and  colon  bacilli.  4  to  5  hours  growth. 

tWright  now  heats  tlie  bacillary  mass  to  100  C.  on  three  successive  days  and 
employs  a   1.5   per  cent,   salt   solution. 

12.  In  all  cases  we  have  used  leucocytes  from  supposedly  normal  human  beings, 
but  it  would  seem  from  the  researches  of  other  investigators  that  the  amount  of 
variation  in  the  phagocytic  power  of  leucocytes  from  different  individuals  of  the  same 
species,   is   so  slight  as   to   be  negligible. 


Ill  list  ration  of  the  Method.— U  it  is  desired  to  obtain  tlie  opsonic  index  to 
staphylococci  of  a  patient  siifferino;.  say.  from  acne,  equal  parts  of  washed  cor- 
puscles, bacterial  suspension  and  patient's  serum  arc  mixed  in  the  pipette  and 
placed  in  the  incubator.  In  another  pipette  equal  parts  of  washed  corpuscles, 
bacterial  suspension  and  normal  serum  are  likewise  mixed  and  incubated.  At  the 
end  of  15  minutes  smears  are  made  from  each  specimen,  fixed  and  stained.  The 
number  of  staphylococci  per  leucocyte  is  obtained  in  each  case  by  countin-r  the 
cocci  in  the  first  50  polymorphonuclear  neutrophiles  encountered  and  makin<,'  an 
averajje.  If  the  average  for  the  patient's  blood  is  (i  cocci  and  that  for  tlic  normal 
is  8,  the  opsonic  index  to  staphylococci  would  be  G/8  or  0.75. 

FIXING    AND    STAIXIXO. 

For  tubercle  bacilli  our  slides  have  been  fixed  in  formalin  vapor,  stained  in 
anilin  fuchsin.  decolorized  in  acid  alcohol,  washed  in  1/1,000  sodium  carbonate, 
and  counterstained  with  methylene  blue.  For  cocci  and  colon  bacilli  Ave  have  fixed 
and  stained  in  Jenner's  stain,  counterstaining  when  necessary  with  methylene  blue. 

DIFFICULTIES. 

In  a  method  of  this  kind  involving  so  many  steps,  there  are  necessarilv  maiiv 
sources  of  error  and  a  slip  in  technic  at  any  point  Avill  often  invalidate  several 
hours'  work.  A  brief  mention  of  some  of  the  difficulties  and  sources  of  error 
met  with  by  us  may  prove  useful.  It  is  very  important  that  the  bacterial 
emulsions  be  uniform',  free  from  clumps,  and  of  the  right  concentration;  if  too 
thick,  the  bacteria  can  not  be  counted  accurately,  and  if  too  thin,  there  will  not 
lie  enough  to  strike  a  proper  average.  For  cocci,  counts  averaging  5  to  12.  and 
for  bacilli,  3  to  5.  are  ideal." 

The  greatest  difllculty  occurs  with  tubercle  bacilli,  since  on  artificial  media 
they  grow  only  in  conglomerated  masses.  For  this  reason  they  have  to  be 
ground  up  in  the  mortar;  but  there  is  an  added  difficulty  from  "the  fact  that 
even  after  they  are  mechanically  separated  they  are  clumped  by  salt  solution  of 
any  concentration  greater  than  0.1  per  cent.,  and  living  bacilli,  furthermore,  are 
agglutinated  by  any  blood  serum.  To  obviate  these  difficulties,  the  bacilli  are 
heated  at  100  C.  for  ten  to  fifteen  minutes  and  the  suspension  is  made  in  0.1  per 
cent,  salt  solution.!  In  using  cocci  and  colon  bacilli  it  is  necessary  to  use  youn^ 
cultures,  since  the  dead  and  dying  cocci  stain  very  poorly  and  irregularly.  * 

Another  source  of  error  in  making  a  series  of  tests  is  dependent  on  the  fact 
that  the  number  of  bacteria  ingested  by  the  leucocytes  during  incubation  varies 
directly  as  the  time  which  the  leucocytes  have  to  act.  This  is  shown  very  clearly 
by  tlie  following  table: 

l-:i'I'K(T     l;I-     'I'lMi;     OF     iNCUn.VTIOX     ON     THE     A.MOrXT     OF     PlI  Afil  )C  VIMS  IS. 

Xuinl)er  of  slai)livloc(K-(i 
Inciilwtion  liiiK"  per  loucdcytV. 

."    iniiiures    _-,  - 

111    in  in  u  res     s;  ;_> 

I"    minutes    1  l.ij 

liii    minutes ]4  - 

L'.'i    miiinles    ].-,■> 

4.">    minuics    I H  4 

1    liDur    I's  .■> 

-    lioiii's    Too    ninn.v    Ik   (■cnnl 

For  slight   (lillcreiices   in  time,  liowcvci-.  tlie  error  is  fortunately  a  >iiiall  one. 

For  the  sake  of  accuracy  the  capillary  i)ii)ettes  should  be  approximately  of 
the  same  caliber. 


1;'..  A  scale  of  opalescence  hy  wliicli  to  .iudme  of  tlie  thickness  of  the  liaiterial  sus- 
pension can  be  made  by  having  a  series  of  liiljes  of  barium  sulphate  of  (iitleieiit  con- 
centrations.      (McFarland.) 


The  '•leupoc-ytic  frcnin"  of  tlio  wasliod  forpusclcs  sliould  bo  used  fn-sli,  a>  ot'iu'v- 
wisp  the  leiieoeytcs  stitk  tofrothor.  and  it  should  he  as  tliick  as  possible  in  order 
to  make  good  smears. 

SMKARS    AXU    COL'XTING. 

Smears  are  made  best  by  iising  a  slide  with  eorner  broken  off  and  the  end 
made  rough  with  a  file.  They  should  be  fairly  thick  and  are  more  easily  made  by 
pushing  the  slide.§ 

In  counting,  the  edges  of  the  smear  are  selected  as  most  of  the  leucocytes  col- 
lect there  and  at  the  end,  and  it  is  better  to  take  isolated  leucocytes  or  at  most 
groups  of  two  or  three,  as  the  counts  for  these  are  more  uniform  than  when  large 
masses  of  leucocj-tes  are  enumerated.  ^Ye  have  found  it  necessary  to  count  a 
larger  number  of  leucocytes  than  did  Wright  and  Douglas  in  order  to  get  a  fair 
average,  consequently  we  have  usually  counted  100  leucocytes  in  working  with  the 
tubercle  bacillus,  and  50  to  60  when  using  the  cocci  and  colon  bacillus. 

The  time  that  it  takes  to  complete  a  set  of  observations  depends  largely  on  the 
worker,  but  after  the  bloods  are  collected  and  the  tubes  opened,  one  should  in  an 
afternoon  be  able  to  make  all  preparations  and  make  slides  of  fifteen  test  sera, 
using  each  to  two  different  bacteria. 

The  enumeration  takes  time  and  depends  very  much  on  the  quality  of  the 
smears.  With  practice  one  can  count  on  an  average  about  five  slides  an  hour, 
sometimes  more,  but  often  less.  To  make  an  index  of  two  or  three  sera  to  a  single 
germ  should  not  take  more  than  two  hours   ( two  workers ) . 

XATURE   OF    OrSOXIXS. 

Eehition  io  Amboceptors. — Savtclienko"  and  Dean^-''  regard  certain 
opsonins  and  amboceptors  as  identical.  Hektoen^"  regards  opsonins  as 
distinct  from  amboceptors,  and  in  proof  of  his  opinion  states  that  nnder 
certain  circumstances  normal  serum  may  possess  lytic,  but  not  opsonic 
powers,  and  vice  versa;  again,  that  immunization  may  give  rise  to 
opsonic,  but  not  to  lytic  substances ;  and,  further,  that  heat  may  destroy 
tlie  opsonic  power  without  affecting  the  lytic  amboceptors  and  vice  versa. 
Tbus.  while  opsonin  for  anthrax  bacilli,  present  in  the  serum  of  normal 
dogs,  is  destroyed  by  heating  at  G0°  C.  for  thirty  minutes,  the  amboceptor 
for  anthrax  bacilli,  present  in  the  serum  of  normal  dogs,  is  not  affected 
by  heating  at  65°  C.  for  thirty  minutes.  Moreover,  while  the  serum  of 
\vhite  rats  is  normally  anthracidal  owing  to  the  presence  of  a  thermo- 
stabile  substance  that  is  inactivated  l)y  neutralization  of  the  serum  with 
oxalic  acid,  the  same  senmi  contains  a  thermostabile  opsonin  for  anthrax 
bacilli,  which,  however,  is  not  inactivated  by  oxalic  acid. 

Relation  of  Opsonins  to  To.rins  and  Complements. — Walker's^'  ex- 
periments with  diphtheria  bacilli  and  tubercle  bacilli  showed  that  toxins 
as  well  as  opsonins  produce  an  effect  on  phagocytosis,  but  that  thev  act 

§Wright  rubs  the  slides  with  a  very  fine  emery  paper  obtained  at  a  jewlei's  and 
uses  a  smooth   smearer. 

14.  Ann.   de  I'lnstitut  Pasteur.   1902.  xvi,   p.   107. 

15.  Proc.   Royal    Soc,   1905.   Ixxvi,   p.   506. 

16.  The  .Tot-kxal  A.   M.  A..  May  12.  1906.  p.  1411. 

17.  "The  Relative  Influence  of  Blood  Fluids  and  Bacterial  Toxins  on   Phagocytosis," 
.Tour.   Med.   Research.   Xov..  1905,  p.   173. 


intlepenclontly  of  one  auotlior.  Opsonins  may  be  considered  analogous 
to  toxins  and  complements  in  so  far  as  they  have  two  distinct  groups, 
Jiaptophore  and  opsoniforc.  Like  complements,  opsonins  may  be  neu- 
tralized or  bound  by  various  salt  solutions,  Ca,  Ba,  MgCL,  K^SO^, 
XaHCOa,  etc.,  and  so  prevented  from  acting  on  bacteria. 

Relation  of  Opsonins  to  " Stimulins." — In  his  treatise  on  "Immun- 
ity/' Metchnikoff  described  a  series  of  experiments  conducted  by  himself, 
Gengou,  Klemperer,  Besredka,  and  others,  in  which  the  introduction  of 
serum,  sometimes  normal,  sometimes  immune,  into  the  serum  sacs  of 
experimental  animals  demonstrated  a  greatly  enhanced  phagocytic  ac- 
tivity in  the  leucocytes  of  the  exudation  subsequently  removed  from  the 
sac  for  examination.  To  the  substance  which  these  observers  conceived 
to  l)e  present  in  such  sera  they  gave  the  name  "stimulin,"  and  to  the 
presence  of  these  "stimulins"  in  immune  sera  Metchnikoff  and  his  school 
attril)uted  an  important  role  in  the  processes  of  both  active  and  passive 
immunization. 

Wright  and  Douglas"  endeavored  to  determine  whether  unhealed 
serum  contained  "stimulins"  in  addition  to  opsonins.  They  found  the 
same  difference  between  the  action  of  heated  and  unheated  serum  toward 
staphylococci  heated  to  115°  C.  as  toward  living  germs.  Their  expecta- 
tions were  based  on  the  fact  that  typhoid  bacilli  heated  above  70°  C. 
acquire  a  resistance  to  bacteriolytic  eifect.  Although  their  experiments 
witli  particles  of  India  ink  and  carmine  wore  unsatisfactory,  unheated 
serum  seemed  to  facilitate  their  phagocytosis  slightly  more  effectually 
than  heated  serum.  Staphylococci  and  unheated  serum  were  digested  in 
an  incubator  for  varying  times  and  then  divided  into  two  portions.  The 
first  was  immediately  mixed  with  corpuscles,  the  second  was  first  heated 
to  60°  C.  and  then  mixed.  In  every  case  the  phagocytic  power  was  greater 
when  the  heating  was  omitted  and  no  difference  could  be  detected  in 
specimens  left  in  the  incubator  fifteen  minutes  from  those  left  one  hour; 
thus  they  were  obliged  to  leave  the  question  unsolved. 

In  190.5  Leishman^**  showed  that  sera  derived  fi'om  cases  of  Malta 
fever  or  enteric  fever,  or  from  animals  immunized  with  living  cultures 
of  the  germs  of  these  diseases,  contain  substances  which  when  added  to 
normal  human  blood  are  capable  of  increasing  the  phagocytic  activity  of 
the  leucocytes  of  that  blood.  He  also  showed  that  these  substance?  were 
specific  and  thermostabile,  withstanding  a  temperature  of  60°  C.  for  fif- 
teen minutes.  No  evidence  of  the  presence  of  these  substances  could  be 
found  in  the  sera  of  nomial  men  or  animals.  He  also  showed  that 
these  substances  were  not  identical  with  agglutinins,  that  llioy  acted 
directly  on  the  leucocytes  and  not  on  the  bacilli.     ^Vri'tchuikofl'  suggested 


IS.   "Some   Experinn'iits    in    ConnofUon    wilh    Stimiilii)s,"    Trans.    I'atli.    Snc.    Kondon. 
1005,   Ivi,   p.    .344. 


the  naiiii'  '•opsonic'  sensitizers"  for  these  siihstances.  W'l'i^dit  sii,2:fjeste(] 
tliat  they  niiiiht  he  tliermostahile  opsonins  in  spite  of  the  fact  that 
opsonins  act  only  on  the  hacteria,  while  these  substances,  if  the  work  and 
statement  of  Leishnian  are  to  be  accepted  as  correct,  act  on  the  leucocytes. 
Effect  on  Opsonins  of  Various  Conditions  and  Agencies. — 1.  Stand- 
ing: The  opsonic  power  of  serum  kept  at  2°  C.  is  practically  unchanged 
in  ten  days,  but  it  is  destroyed  in  three  days  if  the  serum  be  kept  at 
37°  C.  Without  referring  to  the  temperature,  "Wright  and  Douglas  say, 
"Opsonic  power  of  blood  disappears  gradually  on  standing.  In  five  or 
six  days  it  is  only  about  one-half  as  much  as  the  original." 

2.  SiinligJtt:  Exposure  to  l)right  sunlight  for  three  hours  reduces 
opsonic  value  about  one-third. 

3.  Heat:  The  opsonic  power  of  any  serum  is  but  little  impaired  l)y 
heat  below  50°  C.  but  that  of  normal  serum  is  rapidly  destroyed  at  tem- 
perature of  60°  C.  or  above.  ISTormal  human  serum  contains  an  opsonin 
for  typhoid  bacilli  that  resists  heating  aljovo  60°  C,  and  there  is  an 
opsonin  for  anthrax  bacilli  in  the  serum  of  white  rats  that  resists  heating 
to  70°  C.  Dean^'  believes  that  some  of  the  opsonin  is  unaltered  even  by 
heating  to  60°  C,  because  on  exposing  bacteria  to  heated  serum  for  a 
longer  period  than  fifteen  minutes  some  phagocytosis  occurred. 

Immune  opsonins  are  more  resistant  than  the  nonnal.  Leishman^-'' 
and  his  co-workers  showed  that  the  serum  of  persons  vaccinated  against 
typhoid  fever  contained  sufficient  opsonins  to  promote  some  degree  of 
phagocytosis  after  heating  to  56°  C.  Hektoen^''  mentions  that  he  has 
several  times  noted  a  typho-opsonin  in  the  serum  of  convalescents  from 
typhoid  wdiich  resists  heating  to  70°  C.  for  thirty  minutes.  Immune 
hem-opsonins  are  also  thermostal)ile. 

Dilution :  The  opsonic  power  of  serum  is  diminished  in  proportion 
to  the  dilution.  Simon-''  has  utilized  this  j^rinciple  in  estimating  the 
opsonic  content  of  serum. 

5.  Chemicals  and  Drugs:  Hektoen  and  Euediger-'^  showed  that  the 
opsonic  action  of  serum  may  be  diminished  or  inhibited  by  solutions  iso- 
tonic with  the  serum  of  CaCL,  BaCL,  SrCl.,  MgCL.,  K^SO^,  XaHCO,, 
KagHjCgOT,  jSTaoCoO^,  K^re(CX)g,  formalin,  lactic  acid  and  chloro- 
form. They  suggest  that  certain  so-called  negatively  chemotactic  sul)- 
stances,  of  which  lactic  acid  is  a  good  example,  owe  their  effect  to 
neutralization  or  destruction  of  the  opsonin.  The  diminution  of  resist- 
ance produced  by  lactic  acid  to  various  infections  and  ascribed  to  its 
direct  repulsion  of  phagocytes  is,  therefore,  perhaps  dependent  primarily 

19.  Jour,   of  Hyg..   lOO.".,   v.   380. 

20.  Johns   Hopkins   Hosp.    Rep.,   Jan..    1000. 

21.  "Studies   in   Pliagooytosis,"   Jour.   Infent.   Dis..   1005.    vol.    ii.    pp.    ]-J'.t.    141. 


on  tlu'  anti-opsonif  action  of  tlic  acid.  'I'lic  addition  of  potassium  iodid 
in  small  amounts  of  serum  does  not  intei-rere  with  its  opsonic  power." 

Tlic  administration  of  potassium  iodid  has  no  influence  on  the 
opsonic  index.  The  iodid  eruption  is  not  associated  with  a  low  opsonic 
index.  Owing  to  the  fact  that  nuclein  has  a  bactericidal  action  and 
may  cause  leucocytosis  when  introduced  into  the  blood  stream,  the  idea 
occurred  to  Huggard  and  Morland,-^  in  1905,  to  determine  whether  thesa 
manifestations  were  also  accompanied  by  any  change  in  the  opsonic  index 
after  the  employment  of  nuclein  or  yeast. 

After  a  single  dose  of  3  gm.  of  yeast  the  opsonic  index  dropped  for 
two  davs  and  then  rose  some  distance  above  the  normal.  As  has  been 
noted,  this  rise  in  opsonic  index  occurred  at  the  time  when  the  leucocy- 
tosis was  diminishing  and  below  normal.  Huggard  and  Morland  also 
made  the  observation  that  some  of  the  lowest  opsonic  indices  in  cases  of 
tuberculosis  under  their  observation  occurred  in  heavy  smokers. 

6.  Daboia  Venom:  The  opsonic  index  is  diminished  on  digestion  of 
serum  with  daboia  venom. "^ 

RELATION^  OP  OPSOXIXS  TO  LEUCOCYTOSIS. 

From  the  fact  that  leucocytosis  is  of  almost  universal  occurrence  in 
the  course  of  infections,  it  might  be  thought  that  some  relationship 
existed  between  the  quantity  and  equality  of  the  leucocytosis  and  tb.e 
opsonic  content  of  the  serum. 

In  their  research  on  the  therapeutic  effect  of  the  administration  oi' 
yeast  in  tuberculosis,  Huggard  and  Morland-^  found  that,  after  a  pre- 
liminary rise,  the  number  of  leucocytes  remained  constantly  below  nor- 
mal. As  the  opsonic  index  in  these  cases  was  at  first  low  and  later  high, 
it  was  apparent  that  there  was  no  direct  relationship  here  between 
leucocytosis  and  opsonic  index.  The  same  results  were  obtained  by 
Bulloch  and  Ledingham.-*  They  employed  tallianin  and  sodium  cinna- 
mylicum,  both  of  which  increased  the  number  of  leucocytes  without 
increasing  the  opsonic  index,  thereby  showing  also  the  lack  of  relation- 
ship between  the  two. 

SOURCE   OF   OPSONINS. 

Up  to  the  present  time  very  little  has  been  determined  concerning  the 
source  of  tlie  opsonins,  but  Denys  and  Leclef,  AVright  and  Douglas,  and 
Xeufeld  and  IJiiiipau  have  all  shown  that  the  opsonin  exists  in  the  blood 
serum  and  not  in  the  leucocvtes. 


'2'1.  Western  :  "Influence  of  Potassium  Iodid  on  Kesistanoe  of  RIood  Fluids  to 
Staiihylococeus    Albus,"    Brit.    .Tour,    of    Dermatology.    irtO.">,    August. 

'_'."?.  "Action  of  Yeast  in  Tuberculosis  and  Its  Influence  on  the  Opsonic  Index," 
Lancet,   June  :i,   UKt.".   p.   141>:?. 

24.   "Treatment   of  Tuberculosis  by   Tuberculin,"   Lancet,   Dec.   21,   1005,   p.   1C03. 


Tlic  lack  of  any  relation  between  opsonins  and  leucocytosis  might  be 
regarded  as  some  evidence  against  Metcbnikoff's  belief  that  the  leucocytes 
themselves  furnish  opsonin  to  the  senmi. 

RELATIONT    OF    VIKULEXCE    OF    J5ACTERTA    TO    OPSOXIXS. 

Denys  and  Leclef-^  (1905),  Ilektoen  and  l^uediger-*  (1005)  and 
Hektoen  and  Horton^"  (1906)  have  confirmed  Metchnikoft's  funda- 
mental conception  that  the  increase  of  virulence  of  a  certain  organism 
goes  hand  in  hand  with  its  resistance  to  phagocytosis.  They  worked  with 
streptococci,  staphylococci,  pneumococci  and  anthrax  bacilli. 

Hektoen^"  shows  the  effects  of  different  degrees  of  virulence  in  the 
following  way :  Virulent  organisms,  freed  from  anti-opsonic  substances 
by  washing,  on  being  heated  at  65°  C.  for  thirty  minutes,  are  not  opsoni- 
fied  when  treated  with  nonnal  serum.  Similar  treatment  of  the  avirulent 
strains  has  no  effect  on  their  phagocytability.  Virulent  streptococci 
which  are  not  rendered  phagocytable  for  rabbit  and  guinea-pig  leucocytes 
bv  normal  serum  (rabbit,  guinea-pig,  human)  are  taken  up  to  some 
extent  by  human  leucocytes  after  treatment  with  human  serum.  It  is 
also  kno^\^l  that  virulent  cocci  are  subjected  to  phagocytosis  under  the 
influence  of  immune  serum,  and,  further,  that  immunization  of  animals 
with  virulent  cocci  increases  the  opsonin  in  the  serum.  From  their  work, 
consequently,  it  may  be  concluded  that  the  insusceptibility  of  virulent 
cocci  to  phagocytosis  probably  does  not  depend  on  any  lack  of  affinity 
for  the  opsonin,  but  rather  it  would  seem  on  increased  resistance  to  its 
peculiar  action.  Avirulent  bacteria  also  probably  differ  from  the  non- 
virulent  in  producing  substances  which  are  harmful  to  the  phagocytes. 

Helctoen  suggests  that  the  relative  susceptibility  of  various  bacteria 
to  phagocytosis  under  the  influence  of  normal  serum  may  be  quite  an 
accurate  index,  of  which  practical  use  might  be  made,  of  virulence. 

SPECIFICITY   OF   OPSOXIXS. 

The  results  obtained  by  AVright  and  Douglas  and  by  Bulloch'-"  indi- 
cate the  specificity  of  the  opsonins,  but  to  us  the  question  seems  as  yet 
unsettled.  Wright  and  Douglas"  assume  that  a  high  degree  of  specificity 
exists,  though  their  experiments,  in  which  there  was  a  decrease  in  the 
staphylococcic-opsonic  index  of  serum  to  which  typhoid  Ijacilli  had  been 
added,  would  suggest  that  different  bacterial  opsonins  possessed  part  of 
their  power  in  common.  Bulloch-"  bases  his  conclusions  on  the  follow- 
ing experiments:  Serum  after  being  digested  at  37°  C.  with  staphylococci 
was  freed  from  the  bacteria  by  centrifugalization,  when  it  was  found  that 
the  supernatant,  clear  serum  had  lost  all  opsonic  power  for  staphylococci, 


25.  Cent.  f.  Bakt..  1898.  vol.  xxiv.  p.  GSo. 
•2G.   Lancet,  Dec.  2,  190.">,  p.  1603. 


10 

but  '"■largely  retained"  the  opsonins  for  tiilxTcle  l)acilli:  and,  conversely, 
serum  digested  at  37°  C.  with  tuhercle  bacilli  and  cleared  by  centrifu- 
galization  lost  its  tuberculo-opsonic  power,  hut  retained  that  for  staphy- 
lococci. Simon,  using  his  dihition  method  for  estimating  phagocytosis, 
has  repeated  these  experiments  a  number  of  times,  and  in  every  case 
the  opsonic  power  for  staphylococci  and  for  colon  bacilli  declined  in 
about  the  same  ratio. 

Our  experiments  also  give  opposite  results  from  those  of  Bulloch, 
Xormal  "pooled"  serum  from  four  healthy  men  was  used  in  our  test. 
Tliis  was  divided  into  three  parts  and  placed  in  tuVjes.  In  one  tul)e 
stapliylococci  were  thoroughly  mixed  with  the  serum,  in  another  colon 
bacilli  were  mixed  with  the  serum,  and  the  third  tul)e  was  used  unal- 
tered as  a  control.  These  were  placed  in  the  incubator  at  37.. 5  C.  for 
forty-five  minutes,  and  at  the  end  of  that  time  were  subjected  to  cen- 
trifugalization  at  high  speed  for  thirty  to  forty  minutes.  The  serum 
digested  with  stajihylococci  showed  an  opsonic  index  (the  control  tube 
being  taken  as  1.0)  against  staiihylococci  of  0.55,  against  colon,  0.12. 
The  serum  digested  with  colon  l^acilli  gave  an  index  against  stapliylococci 
of  0.26,  but  against  colon  of  lA. 

The  high  figures  for  the  staphylococcic-opsonic  index  of  the  serum 
digested  with  staphylococci  (0.55)  and  for  the  colon-ojjsonic  index  of 
serum  digested  with  colon  bacilli  (1.4)  are  without  doubt  due  to  the  fact 
that  the  bacteria  were  not  thrown  down  completely  by  centrifugaliza- 
tion;  but  the  results  are  given,  as  they  show  that  staphylococci  take 
from  serum  not  only  the  opsonin  for  staphylococci.  InU  also  that  for 
colon,  and  vice  versa. 

XOX-BACTEKIAL   OPSONIXS. 

Opsonins  for  other  cells  than  bacteria  have  been  described.  Hektoen^'"' 
found  opsonins  for  blastomycetes  and  probably  trypanosomes.  Barrat'-' 
and  Hektoen  have  observed  the  presence  of  hem-opsonins  in  the  sera  of 
normal  animals.  We,  working  with  human  sera,  have  observed  it  twice 
in  the  serum  of  colored  people  with  tuberculous  lesions. 

It  is  now  well  known  that  there  are  different  kinds  of  substances 
inimical  to  bacteria  and  their  products  in  blood  serum,  e.  g.,  antitoxins, 
agglutinins,  precipitins,  lysins,  and  opsonins.  Antitoxins  neutralize  the 
l^oisonous  effects  of  certain  bacterial  toxins,  the  best  known  being  those 
of  diphtheria,  tetanus,  and  l)otulismus.  When,  however,  we  investigate 
the  blood  fluids  of  animals  inoculated  with  vaccines  and  bacterial  prod- 
ucts other  than  those  mentioned,  it  is  found  impossible  to  demonsti'ate 
the  existence  of  antitoxins,  or,  at  any  rate,  the  quantity  of  antitoxin  is 
so  slight  that  it  eludes  demonstration.     On  studying  the  experimental 


27.  Proc.    Royal    Soc,   H)().'>,   vol.    lx.\v.    p.   r>L'4. 


11 

typhoid  and  cliolera  infections,  rfeiffer  sliowed  that  the  protective  prop- 
erties of  the  imnuine  sera  in  these  diseases  are  due  to  the  presence  of 
lysins.  Experiments  made  in  succeeding  years  have  shown,  however, 
tliat  this  doctrine  of  lysis  is  not  of  universal  application.  The  study  of 
immunity  against  microbes,  like  pneumococcus,  staphylococcus,  strepto- 
coccus, gonococcus  and  tu])erclo  bacillus,  has  led  to  the  greatest  diversity 
of  opinion;  some  claiming  antitoxic  properties,  others  lytic,  and  others 
phagocytic  properties  for  the  different  sera. 

INFLUENCE    OF   VARIOUS    IXFECTIOXS    ON    OPSONINS. 

In  view  of  these  facts,  it  is  of  interest  to  determine  what  infections 
influence  the  opsonin  formation  in  the  l)ody  as  well  as  the  effect  of  vac- 
cines and  antitoxins  on  opsonins. 

The  tuberculo-opsonic  index  in  healthy  individuals  has  been  esti- 
mated by  Bullocli-^  in  a  series  of  thirty-four  medical  students  and  thirty- 
two  nurses.  The  average  for  the  sixty-six  cases  was  0.95  with  a  maxi- 
mum of  1.20  and  a  minimum  of  0.80. 

In  twenty  individuals  Urwick'-^  obtained  an  average  of  1.006.  We 
have  made  eighty-one  observations  on  nineteen  supposedly  normal  per- 
sons and  have  obtained  an  average  opsonic  index  of  0.988  with  a  maxi- 
mum of  1.25  and  a  minimum  of  0.73."'' 

IJrwick-''  also  proved  that  the  tuberculo-opsonic  index  in  healthy 
people  does  not  vary  except  within  narrow  limits  from  day  to  day. 

Our  series  of  nineteen  normals  estimated  during  the  past  three 
months  shows  the  following  variations  in  four  persons:  0.84  to  1.18, 
0.81  to  1.10,  0.88  to  1.10,  and  0.92  to  1.06.  Wright  and  Douglas'  nor- 
mals varied  between  0.85  and  1.2. 

The  tuberculo-opsonic  index  in  different  varieties  of  tuberculosis  has 
been  studied  by  a  number  of  observers.  Bulloclr^  examined  150  cases 
of  lupus  from  the  mildest  type  to  the  most  chronic  and  intractable  and 
obtained  an  average  index  of  0.75,  the  figures  being  as  follows : 

Opsonic  index 

Between  No.  of  Cases.  Percentage. 

0.2  and  0.3  3  2 

0.3  and  0.4  3  2 

0.4  and  0..5  21  14 

0.5  and  O.G  29  19.0 

0.6  and  0.7  33  22 

0.7  and  0.8  22  14.8 

0.8  and  0.9  18  12 

0.9  and  1.0  7  4.6 

1.0  and  1.4  14  9.3 

Urwick  examined  fifty-four  cases  of  tuberculosis,  including  a  number 
of  cases  of  phthisis  in  various  stages.     In  many  of  them,  especially  of 

28.  "Opsonic  Content  in  Lupus,"   Trans.   Tath.    Soc,   London,   1905,  vol.   xvi,   p.   334. 

29.  Brit.    Med.    .Tour.,    1905,    July    22. 

30.  For  the  material  used  throughout  our  work  we  wish  to  express  our  thanks  to 
the  staffs  of  the  New  York.  St.  Luke's,  Roosevelt,  City,  French,  Ruptured  and  Crip- 
pled, and   Hudson   Street   Hospitals,   and  to   Dr.   Linsly   R.   Williams. 


13 


plitliisis,  the  index  was  al)Ovo  normal,  witli  variations  from  0.3  to  2.6. 
In  thirtv-three  cases  of  phthisis  twenty-five  showed  an  index  al)ove  1.0, 
seven  hehnv  1.0,  and  one  was  at  1.0. 

"Wriiilif  and  Douglas^^  examined  sixteen  eases  of  tuberculosis,  includ- 
ing tuberculosis  of  skin  and  subcutaneous  tissues,  tuberculous  laryngitis, 
cystitis,  peritonitis,  prostatitis,  and  tuberculous  glands.  The  indices 
ranged  from  0.-4  to  0.9. 

We  have  made  observations  on  thirty-nine  cases  of  tuberculosis  as 
follows:  Tuberculosis  of  bones,  including  spine,  hip,  knee,  ankle,  tarsus 
and  tibia,  in  all  22;  above  1.0  in  five  cases,  below  1.0  in  fourteen,  and 
normal  in  three.  Fourteen  cases  of  pulmonary  phthisis  varying  from 
O.-Ll  to  1.37;  "above  1.0  in  one  case,  below^  in  eleven,  and  normal  in  two; 
and  isolated  cases  of  lupus  and  tuberculous  glands  ranging  from  0.96  to 
1.5  J. 

We  have  estimated  the  tuberculo-opsonic  index  in  a  series  of  patients 
with  various  infectious  diseases.     Table  1  shows  our  results.^- 


TABLE    1. 


-Tuberculo-Opsuxic    Index    ix 
sion   of  p.acilli  generally 


Various    Infectious    Diseases. 
IN   1.6  Per  Cent.  NaCl. 


Emul- 


Diagnosis. 


Index. 


hip   and   .spine 1.14 

spine     0.7:-! 

spine    0.92 

spine     1.01 

spine   and  empyema 0.80,  0.8U 

Tb.  empyema  and  peritoneum.  .  .  . 

1.02.  1.02 

knee    0.78 

hip     0.46 

hip,    with   temp l.Od 

hip    with    temp 1.17 

hip.   with   temp 1.13 

hip    with    temp n.Sd 

hip     1..59 

hip.    apvresia o.7."> 

hip     0.86 

hip     0.8.-. 

anlile     O.oO 

anlile    0.7.'),  etc. 

anlile    O.'.to,  etc. 

anl<le     l.O."..  1.18 

Lupus     0.72,  etc. 

TV),     pleurisy     0.89 

Pulmonary    tb 1.00,  !.().-> 

tb n..-.i) 

tb 0.9:! 


Tb 
Tb 
Tb 
Tb 
Til 


Tb. 
Tb. 
Tb. 
Tb. 
Tb. 
Tb. 
Tb. 
Tb. 
Tb. 
Tb. 
Tb. 
Tl). 
Tb. 
Tb 


Pulmonary 
Pulmonary 
Pulmonary 
Pulmonary 
I'ulnionary 
Pulmonary 
Pulmonai-y 
Pulmonary 


tb. 
tb. 
tb. 
tb. 
lb. 
tb. 


0. .-)(!.  0.2.-. 

(1.41 

(1.87 

0.91 

o.ss 

0.89 


Diagnosis. 

I'ulnionary    tb 

Pulmonarv    tb 

Tb.    larsus     

Tb.    ghinds.   jaundice    

Intrathoracic    growth    tb.    glands.. 
Sycosis   and   debility.    F.    II.    of  tb. 

As.    tb.   pneumonia    

Ac.   tb.   pneumonia    

Pneumonia     

I'lieumonia      

Pneumonia     

I'neumonia     120, 

]'(]stpniMini<itiia    empyema     

I'ost  i/ni'uniunin    eini)yenia    

I'Dsipiieiinicinia    empyema    

Typhoid     

Typhoid     

Typhoid     

(iono.    arthritis    ].!.■>. 

<Jono.     arthritis     (J. 67,  0.76, 

Gono.     arthritis     

Osteomyelitis    of    tibia     

Osteomyelitis,     non-tb 

Pus    kidney    0.78, 

Paral.    agitans.    cyst    1.38, 

Malig.    endocarditis    

Abscess   of   liver    

Acne     

Acne     

Acne     

,\cne     

Ajjpendicitis      


ndex. 
1.37 
1.00 
0.8.5 
0.96 
1.51 
0.88 
0.84 
0.86 
1.10 
0.92 
1.01 
0.89 
0.84 
0.86 
0.7."'. 
1.01 
0.68 
0.6H 
1.09 
0.79 
0.94 

o.r.i 

0.91 
0.61 
1.38 
1.06 
l.lo 
1.10 
0.86 
1.07 
0.85 
1.04 


In  thirty-six  cases  of  tuberculosis  the  tiil)eiviilo-opsonic  index  is 
below  normal  (0.75)  in  25  per  cent,  of  the  cases,  and  above  normal  (1.2) 
in  8  per  cent,  of  the  cases;  in  other  words,  is  abnormal  in  33  per  cent, 
of  the  cases.     Of  the  cases  which  had  high  indices,  33  per  cent,  were 


31.  "Action  on   Staphylococcus   by   Human   P.lood  Serum,"   Proc.    Koyal   Soc,   .luly   22, 
1904.   vol.    Jxxiv. 

32.  Experiments    performed    in    the    Pathological    Laboratory.    College    of    Physicians 
and   Surgeons,   Columbia  University. 


13 

cases  with  a  systemic  reaction,  and  of  tliosc  cases  wliich  liad  low  indices, 
66  per  cent,  had  strictly  local  lesions. 

Of  twenty-tliree  cases  with  infections  other  than  with  the  tubercle 
bacillus,  the  tuberculo-opsonic  index  was  below  normal  in  26  per  cent., 
and  above  normal  in  4  per  cent,  of  the  cases. 

EFFECT  OF  OTHER  DISEASES  OX  OPSONINS. 

The  effect  which  certain  chemicals,  of  which  lactic  acid  is  an  example, 
have  on  the  opsonic  action  of  serum  has  already  been  noted,  and  suggests 
the  possibility  that  non-specific  anti-opsonins  may  be  important  factors 
in  the  establishment  and  spread  of  various  infections.  It  is  possible  that 
substances  with  anti-opsonic  action  arise  in  consequence  of  local  met- 
abolic disturbances.  Hektoen  suggests  that  perhaps  it  is  in  this  direction 
that  we  should  seek  a  better  understanding  of  the  so-called  vital  resist- 
ance variations  which  we  believe  permit  the  development  of  primarj^  and 
secondary  infections  in  which  phagocytosis  is  an  essential  means  of 
defense. 

It  was  with  an  idea  of  throwing  some  light  on  this  question  that  we 
determined  the  opsonic  indices  of  a  number  of  conditions  of  disturl^ed 
metabolism,  especially  those  of  a  non-infectious  nature,  with  several 
organisms,  with  the  results  shown  in  Table  2. 


TABLE   2. — Orsoxic   Index  ix   Nox-Ixfectious  Diseases. 


Index  to 
Colon. 


1.02 


Index  to       Index  to       Index  to 
Diagnosis.  Tuber.  Staph.  rneumoc. 

Secondary    anemia    0 . 9.5  1 .  09 

Cirrhosis    (.Foiin's    diet)     1.13         \     0.45* 

Hodgkin's    disease     0 . 8.5  1 .  00 

Thoracic    tumor     1.16  0.90 

Icterus    catarrhalis     0.98 

Acute    lymphatic    leukemia    1 .33 

Purpura  :    multiple    sclerosis     0.7.5 

Cardio-nephritls     0.9.5 

Chronic   parenchymatous   nephritis    0.95 

Chronic   parenchymatous  nephritis    0.59* 

Herpes    zoster ;    hydrothorax     1.00 

Chronic    gout     0 .  98 

Chronic    gout     0 .  85 

Paralysis    agitans    (cystitis)     1.38 

Ringworm    of    body 0. 88 

Fracture    of    hip    0 .  79 

Carcinoma  of  stomach    0.67* 

Ovarian     cyst     1.10 

Hodgkin's    disease     1 .02 

Carcinoma    of    stomach     1 .06  0 .  93  0 .  72  1 .  50 

Carcinoma    of    stomach     .•••  ••••  1.19 

Pleurisy,   .serofibrinous    •  •  •  •  •  •  •  •  0 .  83 

Cardio-renal    0 .  60* 

Cardio-renal     •  •  •  •  •  •  •  •  0 .  66* 

Intrathoracic    new    growth    0 .  90 

♦These  figures  are  starred  to  emphasize  low   Indices. 

At  the  present  time  it  would  be  difficult  to  know  how  to  interpret 
some  of  these  figures.  It  is  evident,  however,  that  some  very  low  as 
well  as  high  indices  may  occur  in  the  absence  of  any  demonstrable  infec- 
tion. While  several  of  the  low  indices  are  obtained  in  conditions  in 
which  there  is  some  interference  with  the  digestive  function,  two  such 
cases  (carcinoma  of  the  stomach)  show  a  comparatively  high  index. 


14 


"We  have  attempted  to  corajDare  the  tuberculo-opsonie,  staphylococcic- 
opsonic,  streptococcic-opsonic,  pneuniococcic-opsonic,  gonococcic-opsonic, 
and  eolon-opsonic  indices  in  a  few  cases.  Our  results  are  exhibited  in 
Table  3. 

TARLE  ."5. — Oi'SdMc  iNDiCKs  IN  Vakious   Infkctious   Diseases  to  Tubercle  Bacilli 

AND  Other  Germs. 

Index    Index    Index   Index    Index    Index 


Case  No. 


Diagnosis. 


to  to  to  to  to 

Tb.    Staphy.  Pneu.  Strept.  Colon. 


to 
Gon. 


0.72* 
(t.ti(5» 


0.72* 


O.n.i 
1.09 


1.12 

o.nir 

0.70 

0.7.-.* 

0.72" 


1  .10 
1  .  20* 


1.  Tb.   hip  and  spine 1.14 

2.  Tb.    hip    l.(Mi 

:i.  Tb.    hip     1.13  0 .  78 

4.  Tb.   hip    with    tenii).    and   abscess 0 . 8.->  1.00 

5.  Tb.    hip    and    sinus     1  ..-.9*  1 .00 

«.  Tb.    knee,    apvre.xia    0.78  0.80 

7.  Tb.    anlcle     .  ." 0.89  0.76 

8.  Tb.    ankle     1.12  1.20* 

9.  Tb.    pulmonaiv    0.91  .... 

10.  Tb.    pulmonary    0 .  87  0 .  90 

11.  Tb.    glands,    jaundice    0.96  .... 

12.  Tb.    glands,    of   mediastiiuim 1  ..->0*  0.90 

13.  I'leurisy    tb.    V    (chest   fluid) 0.78  

(blood    serum ) 0 .  89  .... 

14.  Syphilis     (secondary)      1  .02  0.83 

15.  (ion.    arthritis     1 .  09  1 .20* 

16.  Typhoid     1.01  0.77 

17.  Typhoid     0  .  68*  1 .  l.'j 

18.  Typhoid      0 .  68*  1.07 

19.  Pneumonia     0.92  .... 

20.  Pneumonia      1 .  20*  0 .  .-)4» 

21.  I'neumonia     1 .  (II  0 .  94 

22.  l^neumonia      ]  .10  .... 

23.  Abscess  of  liyer    1 . 06  0 . 93 

24.  Abscess  of  liyer    1.10  0.61* 

25.  Empyema     0 .  75*  .... 

26.  Empyema,    post-pneumonic    0 .  86  .... 

27.  Icterus    catarrhalis     n .  98  .... 

28.  Appendicitis,    post-op 1  .  04  1  .  09      ....      1 .  00 

29.  (^hronic    gon.    arthritis    0.67*  .... 

30.  (^'hronic  gon.   arthritis    1.15  .... 

31.  Tb.    meningitis    0 .  90  .... 

32.  I'ulmonary    tb 0  .  67* 

33.  Empyema    of    gall-bladder 1.02 

34.  Normal      o  .  98  1  .  <I0 

35.  Normal    0.97  1 .  00 

36.  Normal    1 .00  1 .00 

37.  Normal    1 .  07  1  .  00 

38.  Normal    <» . 92  1 . 00 

39.  Normal    110  

40.  Normal    <• . 8.-?  

41.  Normal    1  •  oi  •  0 .  Si 

42.  Normal    1 .  00  1.10 

43.  Normal    <».99  1 .10      1  .  00 

44.  Normal    1 .  05  1 .  00 

45.  Normal    "'  •  81  

46.  Normal    1 . '"O  

47.  Normal    1  .  05  .... 

48.  Normal    !•' -0" 

49.  Normal    '• . 92  

50.  Normal    <•  •  91  1  . 02      

51.  Normal    1  •  02  0 .  98      

♦These  figures  are  starred  to  emphasize  peculiarities  in  indices. 

From  Table  3  it  will  be  seen  that  the  indices  in  a  few  cases  to  dif- 
ferent germs  vary  remarkably,  but  not  as  much  as  one  would  be  led  to 
expect  from  Wright's  work.  Cases  1  and  2  are  worthy  of  note  in  showing 
low  indices  to  staphylococcus  with  normal  figures  for  tubercle  bacilli. 
These  cases  liad  no  sinus  or  staphylococcus  infection.  Case  6,  being 
apyrexial,  should  have  given  a  low  index,  one  would  expect.  Case  20, 
one  of  lobar  pneumonia,  shows  a  low  index  to  stapliylococcus,  a  high 
index  to  tuberculosis,  and  normal  to  ])ncuiii()e(>ctiis.     Case  24  is  the  most 


0.88 


1  .10 
0.91 


1  .(16 


0 .  94 

1 .  06 


1  .  50* 
0.17* 
1  .17 
0 .  83 
1  .  ( 12 
0.74* 


0.89 
0.81 
1.43 


1.10 
0.88 


1  .  03 
1  .04 
0 .  92 
0 . 9,S 
1.01* 


15 

rcmarkal)le  case  in  the  series.  This  patient  liad  an  enormous  al)sces3  of 
tlie  liver  which  had  heen  operated  on  about  a  weelc  l)efore  these  figures 
were  obtained.  Cases  23  and  o'.]  wouhl  al^^o  indicate  a  certain  specificity 
of  tlie  opsonins. 

Staphylococcus. — Wriglit  reported  twenty  cases  of  localized  sta])liy- 
lococcic  infection,  viz.,  furuncles,  acne,  sycosis,  etc.,  witli  indices  varying 
from  0.1  to  0.88,  with  an  average  for  the  twenty  of  0.62.  In  twenty-five 
cases  with  uncomplicated  localized  staphylococcic  lesions,  Bullocli  reports 
that  the  index  was  "below  normal  constantly."  A\'e  have  been  unable  to 
find  a  list  of  indices  of  normal  individuals  to  staphylococci.  AVe  have 
estimated  the  indices  of  ten  normal  persons  to  staphylococcus  and  found 
them  to  range  between  0.82  and  1.20,  the  average  for  nineteen  determi- 
nations being  0.98.^^  Three  cases  were  examined  at  intervals  during 
several  months  and  showed  variations  in  one  case  between  0.84  and  1.20, 
in  another  between  0.85  and  1.10,  and  in  a  third  betw^een  0.90  and  1.02. 

Pneumococcus. — While  Dr.  Eosenow  affirms  that  during  the  course  of 
pneumonia  up  to  and  shortly  after  the  crisis  there  is  probably  no  increase 
in  the  pneumococcic-opsonic  index,  MacdonakP*  has  found  that  at  the 
commencement  of  the  infection,  while  the  temperature  is  in  the  ascend- 
ant, and  during  the  fastigium,  the  opsonic  index  is  below  normal.  With 
the  onset  of  crisis,  there  is  a  striking  rise  in  the  opsonic  content,  the 
index  rising  critically  as  high  as  1 .6. 

Typhoid.  —  In  convalescent  typhoid  fever  patients  a  well-marked 
increase  in  the  opsonin  for  typhoid  bacilli  has  been  noted,  the  index  in 
some  cases  reaching  as  high  as  4.0. 

Streptococcus. — Euediger^"  has  noted  an  increase  in  the  strei^tococcic- 
opsonic  index  of  erysipelas  serum. 

Tables  4,  5,  6  and  7  show  the  effect  of  various  infections  on  the 
opsonic  index  to  staphylococcus,  colon  bacillus,  gonococcus  and  pneumo- 
coccus, the  first  cases  in  the  tables  being  caused  by  the  specific  organism 
employed. 

TABLE    4» — Opsonic    Indices    to    Stai'hylococcus    PyooEXES    Aureus. 

1.  Abscess    of    liver 0.9.3        17.  Typhoid    fever    1.1.'> 

2.  Abscess    of    liver 0.61        18.   Typhoid    fever    I.OT 

.S.   Appendicitis     post-oper 1.09        19.   Cirrhosis,    Folin's    diet n.4.. 

4.  Acne      0.7.5,   1.2  20.  Gonorrheal  arthritis     ...    1.20,   1.01 

5.  Furuncle      0.86  21.  Tubercular  arthritis    0.90 

6.  Dermatitis   vegetans    0.82  22.  Tubercular   hip  and  spine 0.72 

7.  Acne      0.94  23.  Tubercular  hip  and   spine    ....    0.66 

8.  Empyema     0.95  24.  Tubercular  hip   and   spine    ....    0.78 

9.  Empyema    of    gall-bladder 1.02  •2'>.  Tubercular  hip   with   temp,    ab- 

10.  Pleurisy,     serofibrinous     0.93  scess      1.00 

11.  Meningitis    cerebrospinal     1.01  2(>.  Tubercular   hip   with   sinus....  1.00 

12.  Meningitis    cerebrospinal    1.02  27.  Tubercular    knee    0.80 

13.  Meningitis    cerebrospinal    0.84  28.   Tubercular    auUle     0.7tf 

14.  Pneumonia     112  29.   Tubercular    ankle     1.20 

15.  Pneumonia     1.09  .•?().   Pulmonary    tuberculosis    0.90 

16.  Typhoid   fever    0.77  ."1.   I'neumonia     0.9S 


3.S.  The  average  count  for  at  least  three  normal  sera  which  have  shown  very  slight 
variations  was  taken  as  1.00. 

34.   Practitioner,   November.    1905. 


16 

or  those  diseases  in  which  the  staphylococcic-opsonic  indices  were 
below  normal,  two  were  caused  by  staphylococcus  infections  and  three 
occurred  in  other  conditions.  Of  those  in  which  the  indices  were  above 
normal,  one  was  caused  by  staphylococcus  infection  and  two  occurred  in 
other  conditions  (Table  4). 

In  the  twelve  normal  cases  in  which  the  colon-opsonic  index  was  ob- 
tained, the  range  extended  from  0.81  to  1.19.  These  figures  may,  then, 
be  used  to  define  the  normal  limits  of  the  colon-opsonic  index  in  Table 
5  of  diseases  due  to  the  colon  bacillus  and  other  infections  or  diseased 
conditions. 

TABLE  5. — Ofsoxic  Indices  to  Colon  Bacillis. 

1     Abscess    of    liver...    0.17.    0.10,  0.2(5  10.  Tub.    glands,    jaundice    1. 

■2.  Abscess    of   liver    0.46  lio.  Empyema     1.17 

.S.   Abscess    of    liver    1.50  21.  Empyema     0.S3 

4.   Appendicitis     (postop.)      0.7.'5  22.  Osteomyelitis    of    femur O.i.j 

.').   Fecal    fistula     0.32  23.  Diabetes     0.6.J 

0.   Peritonitis    (postop.)     l.OO  24.  Normal     1.10 

7.  (Jallstones      1-1  25.  Normal     0.88 

8.  (iallstones    with    jaundice    ....  l.O.^  26.  Normal      0.81 

0.   (Jallstones      1-08  27.  Normal     l.lo 

10.  Empyema     of     gallbladder .  .  .  .  1.43  28.   Normal  1  no 

11.  pyonephrosis     1-49  20.   Normal  l.oo 

12.  CVstitis  and  paralysis  agitans.  1.10  30.   Normal  l.oo 

13.  Icterus   catarrhalis    102  31.  Normal  0.9 

14.  Typhoid   fever    0.94  32.   Normal  0.82 

15.  Typhoid    fever    1 .06  33.   Normal  1.18 

16.  Abscess   of  spleen    0.73  34.  Normal  1.19 

17.  Empyema    (postpneu.)     0.83  35.  Normal  1.02 

18.  I'ulnionary   tuberculosis    1.06 

Among  the  colon  infections  (Cases  1  to  13,  Table  5)  the  index  is  below 
the  normal  in  four  conditions  in  which  the  liver,  vemiiform  appendix 
and  intestine  were  involved,  and  above  normal  in  three  conditions  in 
which  the  liver,  kidney  and  gall  bladder  were  the  organs  involved.  Six 
of  the  thirteen  cases  were  normal.  Of  the  diseases  due  to  other  causes 
than  colon  infections  the  index  is  below  the  normal  in  three  cases,  one 
case  of  diabetes  and  two  inflammatory  conditions;  but  in  none  is  the 
index  above  normal. 

In  the  four  normal  cases  in  which  the  gonococcic-opsonic  index  was 
obtained,  the  range  extended  from  0,9  to  1,09,  These  figures  may,  then, 
be  u.sed  to  define  the  normal  limits  of  the  gonococcic-opsonic  index  in 
Table  G.  Six  of  these  cases  have  indices  below  the  normal,  though  but 
one  has  an  index  below  0.75  and  three  have  indices  above  1,09, 

TABLE    C. Ol'.SOMC     I.NUICKS     to     OoNOCOCCfS. 

Gonorrheal  epididymitis,  subacute    0.62 

Gonorrheal  epididymitis,  subacute    0.77 

Gonorrheal  epididymitis,   svibacute    0.99 

Gonorrheal  epiilidymitis,   subacute    0.87 

Gonorrheal  epididymitis,  subacute    0.88 

Gonorrheal  epididymitis,  subacute    1-01 

Gonorrheal  urethritis,  subacute,  5   days    0.97 

Gonorrheal  urethritis,   7   days    '••95 

Gonorrheal  urethritis,    2    months 0.9(1 

Gonorrheal  urethril is.  4  months    0.77 

Gonorrheal  urethritis,  4   months    l-0."> 

Gonori-heal  urethritis,   ('»  weeks    0.83 

Gonorrheal  ui'ethritis,  8  months    1.23 

Gonorrheal  arthritis    1-23 

<Tonorrheal  arthi-iUs    110 

(Jonorrheal  artluitis    109 


17 

In  Table  7  the  normal  range  in  I'our  eases  was  between  0.88  and  1.15. 
'^^j'wo  cases  of  pneumonia  and  one  oi'  pulmonary  tuberculosis  were  slightly 
below  the  lower  limit  of  the  normal. 

TABLE  7. — Orsoxic  Indices  to  I'nkumococccs. 

Acute   lobar   pneumonia    1.12 

Acute  lobar   pneumonia    O.'m; 

Acute  lobar  pneumonia,  eleventh  day    II"' 

Acute   lobar  pneumonia,   seventh   day    o.T.") 

Pulmonary    tuberculosis    0.72 

I'ulmonary    tuberculosis    l-'»<> 

Tubercular    pleurisy     1-00 

Normal     0.88 

Normal     1-1 

Normal      1-15 

Normal     0-91 

INFLUENCE  OF  ANTITOXINS  ON  OFSONINS. 

The  varied  influence  of  chemicals  and  drugs  on  opsonins  has  already 
been  mentioned  and  helps  to  explain  the  different  effects  on  opsonins 
and  phagocytic  action  of  toxins  of  which  diphtheria  and  tuberculosis 
toxins  may  be  taken  as  examples.  E.  L.  Walker-'*'  has  shown  that,  while 
diphtheria  bacilli  are  less  susceptible  to  phagocytosis  by  human  blood 
after  destruction  of  the  toxins  by  heating  from  90°  to  100°  C,  tubercle 
bacilli  are  more  susceptible  to  phagocytosis  after  destruction  of  the  tox- 
albumins  by  heating  to  from  80°  to  100°  C. 

Another  interesting  factor,  and  one  which  needs  to  be  further  devel- 
oped, is  the  influence  of  antitoxins  on  opsonins.  Bradshaw  and  Glynn'' 
have  recently  reported  nine  cases  of  normal  men  who  had  received  diph- 
theria antitoxin  and  whose  indices  to  tubercle  bacilli  averaged  only  0.73. 
In  one  the  index  was  0.47  after  three  months.  Walker-'^  has  shown  that 
diphtheria  antitoxin  will  apparently  not  neutralize  these  affinities  of  the 
toxin  of  the  diphtheria  bacillus  which  stimulate  phagocytosis. 

Bordet,  Aronson,  v,  Lingelsheim  and  others  have  noted  the  greatly 
increased  phagocytosis  of  streptococci  in  the  presence  of  antistreptococcus 
serum.  At  present  the  principal  serum  used  for  therapeutic  purposes  in 
infections  in  the  healing  of  which  phagocytic  immunity  may  be  regarded 
as  an  essential  phenomenon,  is  antistreptococcus  serum.  Hektoen  affirms 
that  in  tests'  made  a  year  ago  by  Dr.  Euediger  and  himself  the  anti- 
streptococcus sera  then  in  general  use  in  this  country  were  all  shown  to 
possess  a  lower  opsonic  index  for  various  streptococci  than  normal  horse 
serum,  many  samples  being  without  any  opsonic  power,  presumably  on 
account  of  their  age.  Eesults  of  a  similar  nature  were  obtained  by  us 
with  a  specimen  of  antipneumococcus  serum  which  was  five  days  old, 
and  also  with  an  antigonococcus  serum  sixteen  days  old,  which  proved  to 
have  the  most  marked  beneficial  therapeutic  efl^ect.'®     It  is  difficult  to 


35.  Liverpool  Med.  and  Chir.  Jour.,  May.   190G. 

36.  We  desire  to  express  our  thanks  to  Dr.   Torrey.   Cornell  University   Medical   Col- 
lege, for  the  antigonococcus  sera  employed  and  for   numerous   cultures. 


18 

sa}'  wliether  this  was  due  to  tlie  age  of  the  serum  or  to  the  fact  that 
gonococci  do  not  ext-ito  the  formation  of  opsonins  in  immune  sera.  Our 
results,  indicating  the  effect  of  age  on  immune  sera,  are  sliown  in  Table  8. 

TABLE  8. — Effect  of  Age  on   Immune   Sei!a. 

Anti-pnpumoc.   sei-um,   rabbit :  Indox. 

Scrum   5  days  olO    <•.<> 

Serum    fresh    1  ■<> 

Auti-diphther.   serum.   Board   of   Health    serum     (old) 0.4* 

Anti-gonoc.   serum,  rfibbit  : 

Hi  days   old    0.07* 

30   days   old    0.17* 

Goat   12  days   old    0.00* 

♦Normal  human  serum  taken  at  1.0. 

These  results  would  tend  to  show  the  importance  of  using  sera  which 
may  depend  for  their  action  on  their  opsonic  content,  while  the}'  are  still 
veiy  fresh.  It  is  more  than  possible  that  in  infections  in  which  leucocy- 
tosis  is  marked,  such  as  pneumonia,  streptococcus  and  staphylococcus 
infections,  the  failure  hitherto  to  ol^tain  the  desired  results  with  the  use 
of  immune  sera  prepared  by  adaptation  against  these  organisms  may  be 
explained  by  the  rapid  diminution  of  opsonins  here  shown. 

INFLUENCE   OF    VACCINES    ON    OPSONINS. 

The  material  which  has  been  employed  for  therapeutic  inoculations  in 
other  than  tuberculous  infections  has  consisted  of  a  culture  of  the  organ- 
ism causing  the  infection,  grown  in  broth  or  on  agar.  In  the  latter  case 
a  suspension  of  the  bacteria  is  made  in  normal  salt  solution.  After  ster- 
ilization by  exposure  to  a  temperature  of  from  G5°  to  75°  C.  for  half  an 
hour  and  the  addition  of  0.5  per  cent,  lysol,  this  material,  which  is  known 
as  the  '"vaccine,"*  is  ready  for  inoculation.  This  suspension  may  be 
standardized  by  determining  the  number  of  bacteria  per  cubic  centimeter 
ofiluid.3^ 

On  the  inoculation  of  any  vaccine  there  is  at  first  a  period  of  dimin- 
ished resistance  to  the  organism  employed.  This  is  shown  by  a  decline  in 
the  antibacterial  pow'er  of  the  blood,  the  so-called  "negative  phase."  Suc- 
ceeding this  there  is  an  inflowing  of  and  consequent  increase  in  the 
protective  elements  of  the  blood.  This  is  the  "positive  phase,"  or  the 
2>eriod  of  increased  resistance.  Most  of  these  protective  elements  rapidly 
disappear,  but  leave  behind  a  more  or  less  permanent  increase  over  the 
original.  Wright  speaks  of  the  whole  sequence  as  the  "law  of  the  ebb 
and  flow  and  reflow  and  subsequent  maintained  high  tide  of  immunity." 
Figure  1,  selected  from  an  article  by  Wright,^^  illustrates  this  "law." 

This  train  of  events  succeeds  inoculation  of  vaccine  sufficient  to  cause 
a  constitutional  disturbance.  When  only  a  small  dose  of  vaccine  is  given, 
the  negative  ])hase  may  be  so  insignificant  and  of  such  short  duration  as 


♦Wright's  nomienclature   and  explanation. 
'.',7.  "Some    New    Procedures   for    the    Kxaminatiou    of    the    Blood,"    Lancet.    .July 
lliOii. 


19 

not  to  1)0  eapahle  of  measurement,  and  the  p(jsitive  phase  will  he  corre- 
spondingly diminished.  On  the  other  hand,  it.'  the  dose  of  vaccine  is  too 
large,  the  negative  phase  will  l)e  unduly  prolonged,  and  a  positive  phase 
may  not  appear  at  all.  The  question  of  dose,  then,  is  of  the  greatest 
importance,  for,  if  too  little  is  employed,  no  therapeutic  effect  will  result. 
On  the  other  hand,  too  large  a  dose  is  dangerous  and  so  lowers  the  resist- 
ance of  the  host  as  to  favor  the  multiplication  of  the  hacteria.  Such 
clangers  with  a  single  inoculation  are,  of  course,  greatly  magnified  by 
repeated  inoculations.  If  a  series  of  such  inoculations  are  attempted  dur- 
ing the  negative  phase  there  will  result  a  further  fall  in  the  protective 
substances  or  a  '•cumulation  in  the  direction  of  the  negative  phase." 
Sooner  or  later  a  point  will  be  reached  when  the  organism  will  cease  to 
respond  to  inoculation,  and  further  doses    of  vaccine  will  not  only  do  no 


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Case  of  a  physician  wlio  had  suffered  from  boils  for  four  years  (Proe.  Roy.  Soc, 
1904-.5.  vol.  xxiv,  p.  1.50).  A.  Inoculated  with  2,000  millions  sterilized  staphylococci 
derived   from    patient.      B.     The   same   repeated. 

good,  but  may  bring  the  content  of  the  blood  in  protective  substances  to 
a  lower  level  than  that  from  which  it  originally  started. 

AVright^®  considers  tliat  the  result  of  the  vaccination  can  not  be  esti- 
mated from  the  character  of  the  constitutional  disturbance,  and  with 
some  reason  urges  the  daily  determination  of  the  opsonic  index  to 
measure  the  effect.  He  advises  the  smallest  possible  initial  dose  sufficient 
to  produce  an}^  negative  phase  and  reinoculation  only  after  the  subsequent 
positive  phase  has  begun  to  diminish. 

CLIXICAL  APPLICATIOX   OF  IXOCULATIOX   OF  VACCIXES. 

Stapliylococcus  Infections. — Wright  has  cited  twenty  cases  of  very 
obstinate  staphylococcus   infection,   including   sycosis,   boils,   acne,   etc.. 


38.   "General    Principles    of    Therapeutic     Inoculation     of    Vaccines     on     Result     of 
Tuberculous  Infections."'  Lancet.  Dec.  2,   1005.   p.   1000. 


20 

wliicli  lie  had  treated  by  vaccination.  Tliese  cases  were  selected  as  being 
particularly  intractable.  Various  medical  and  surgical  measures  had 
previously  been  tried  without  success.  Some  were  apparently  cured  by 
the  vaccination,  all  showed  a  marked  improvement.  He  suggested  the 
possible  utility  of  vaccinating  patients  before  operations,  especially  oper- 
ations on  the  nose  and  mouth,  also  of  thus  treating  gleet,  bronchitis, 
ozena,  bacteriuria,  etc. 

The  following  cases  of  staphylococcus  infections  have  been  treated 
by  the  author  by  the  administration  of  staphylococcus  vaccines : 

H.  M,  Furunculos^is.  Recurring  furunciilosis  of  the  back  of  the  neck :  an 
active  furuncular  process  at  the  time  of  first  observation.  Opsonic  index  then 
.75.  A  vaccine  was  made  from  the  infecting  organism — staphylococcus — and  two 
inoculations  were  given,  the  first  300,000.000  sterile  organisms,  followed  by  an- 
other of  600,000.000  in  five  days.  Opsonic  index,  1.5.  The  fui'uncle  disappeared 
and  the  patient  was  free  from  trouble  for  four  months.  Then  another  furuncle 
appeared.  Opsonic  index,  .7.  An  inoculation  of  300,000.000  sterile  staphylococci 
was  given.  Index  rose  to  1.6  on  the  second  day,  was  1.4  on  the  third  day,  and  on 
the  fifth  day  had  fallen  to  1.  Another  inoculation  of  600.000,000  was  given,  the 
index  rising  in  two  days  to  1.3.  The  furuncle  healed.  No  more  furuncles  ap- 
peared during  the  subsequent  eight  months. 

Boy  in  Roosevelt  Hospital,  aged  14.  Pyemic  multiple  abscesses.  For  two 
weeks  before  onset  of  trouble  he  and  his  whole  family  had  had  an  epidemic  of 
pimples  and  sores  on  the  skin.  Five  days  before  admission  to  the  hospital  ho 
struck  his  leg;  the  result  was  pain,  swelling,  temperature  of  105.  Incision 
gave  only  temporary  relief.  He  became  delirious.  At  the  hospital  the  compact 
bone  of  the  internal  surface  and  anterior  border  of  the  tibia  was  removed  from 
the  whole  shaft.  The  exposed  bone  was  dark,  soft  and  spongy,  and  cont^iined 
pus  foci.  Irrigation  was  ineffective,  and  at  the  end  of  four  days  the  knee 
became  inflamed  and  full  of  pus;  the  leg  was  amputated.  Staphylococcus  pyo- 
genes aureus  was  found  in  the  blood  at  this  time.  A  focus  of  pus  over  the 
malleolus  was  also  opened  and  drained.  Eleven  days  later  a  focus  on  the  left 
arm  was  opened,  and  during  the  next  week  two  more  foci  were  opened.  Then 
there  occurred  symptoms  of  abdominal  inflammation,  which  were  not  operated 
upon.  Several  more  foci  opened  during  the  next  month.  The  fibula  was  then 
resected.  Sutures  broke  down.  Much  discharge.  On  November  27.  after  four- 
teen weeks  of  illness,  with  patient's  condition  one  of  chronic  multiple  pyemic 
abscesses  without  tendency  to  heal,  inoculations  were  begun  with  a  vaccine  made 
from   his    own    staphylococcus. 

Condition  of  wounds  at  time  inoculations  were  begun  were  as  follows: 

Leg — sinus — eight  inches  long  along  fibula.  Left  arm — small  amount  of 
discharge.  Right  elbow — considerable  discharge.  Abscesses  in  hands.  Opsonic 
index,  November  27  —  0.5.     Three  hundred  million  sterile  staphylococci  injected. 

After  these  inoculations,  the  condition  was  as  follows:  Right  leg— healed. 
Left  arm— healed.  Abscesses  in  hand— healed.  General  condition  markedly 
improved. 

Two  cases  of  staphylococcus  pyemia  secondary  to  a  local  cellulitis 
were  seen  and  inoculated  by  the  author,  four  and  three  days  before  death, 
respectively.  Each  received  400,000,000  sterile  staphylococci.  Both  had 
Ijeen  running  for  days  a  high  pyemic  temperature,  both  had  countless 
subcutaneous  abscesses  with  some  pulmonary  involvement,  and  neither 
sliowcd  anv  iK'udiciiil  vihvi  I'vom  llio  inooulntion. 


31 

Colon  Bacillus  Infections. — Wright  and  EcicF'^  reported  two  cases  of 
cholecystitis  with  persistent  sinuses  after  operation.  Both  were  inocu- 
lated with  sterilized  cultures  of  coll  communis,  and  as  a  result  of  the 
inoculation  the  opsonic  index  rose  above  normal,  in  one  case  to  4.0,  and 
both  soon  showed  marked  improvement,  with  closure  of  the  sinuses. 


Nov.    28     Opsonic    index 1.3 

Dec.       1  "  "... 


10 
12 
13 
14 
15 
16 
17 
18 
1!> 


6 
2.6 
1.3 
1.7 

.7 
2.0 
1.0 

.8 
1.3 
1.4 
1.7 
1.9 

i.r. 

1.8 


300,000,000    S. 

300,000,000 

600,000,000 

600,000,000 


P.    A.    injected. 


Case   M.    II. 


The  following  case  of  infection  by  the  Bacillus  coli  communis  was 
treated  by  the  author: 

M.  R. — On  night  of  admission  to  the  Roosevelt  Hospital,  the  patient  had  been 
caught  between  the  shaft  and  elevator.  The  compression  was  greatest  about  the 
hips.  She  immediately  became  unconscious.  When  her  senses  returned  she  had 
great  pain  in  her  abdomen  and  legs  and  a  feeling  of  numbness  and  helplessness. 
8he  was  brought  to  the  Roosevelt  Hospital  shortly  after  regaining  consciousness. 
X-ray  showed  a  fracture  of  the  pelvis. 

The  patient  was  admitted  to  the  hospital  December  25,  with  a  temperature  of 
96.4.  During  the  next  week  the  temperature  rose  steadily.  A  pelvic  abscess 
formed  pointing  at  a  point  to  the  left  of  the  labia  niajora.  The  patient's  condi- 
tion became  critical.  She  was  operated  on  January  4,  abscess  opened  and  a 
sinus  was  found  communicating  with  a  large  abscess  cavity.     About  four  ounces 


39.  "Note  on  Two  Cases  of  Cholecystitis  Tr«»ated  with  Inoculation  of  Bacillus 
Coli,"  and  "Note  on  the  Phagocytosis  of  Red  Blood  Cells.  Relationship  of  Tempera- 
ture to  Opsonic  Curve  in  Acute  Anthrax  Infection,"  Lancet,  Jan.  20.  1906,  p.   159. 


22 

of  foul  smelling  pus  were  evacuated.  A  piece  of  bone  also  came  out  and  the 
wound  was  drained.  For  one  week  the  temperature  ranged  from  100  to  104 
daily,  leucocvte  count  14,000,  10,000  and  then  21.000.  Pulse  increased  from  IIG 
to   130.   and"  the  patient's  condition  became   so  much   worse  as  to  be  considered 


Month     |4Ur*^^4^'^LA*«l-nJU<r. 


t^,cib^i  /ti^  uyixiiuifU 


Case  H. 


almost  liopcless.  From  the  pus  a  colon  bacillus  had  been  isolated,  and  from  tins 
a  vaccine  was  made.  On  Januarv  0.  25.000.000  sterile  colon  bacilli  were  injected. 
The  temperature  fell  to  101,  and  on  January  12,  50.000.000  sterile  colon  bacilli 
uerc  injected.     A  further  fall  in  temperature  occurred,  the  pulse   rate  becoming 


Month     I *L^,,^^y-y 


Case  II. 

slower  and  H.c  i.atient's  condition  improving.  After  a  third  inoculation,  on 
T-inu'irv  15  of  75,000.000,  a  rise  of  temperature  occurred,  followed  by  a  drop  to 
normal"  this  being  repeated  on  January  10.  From  this  time  on  the  temperature 
r,.n.ai,H.d    normal,   the   pulse  rate  remained  about  90,   and  the  condition   relative 


23 

to  llip   iiifoction   rnpidly  and  pornianciilly   improved,  the  sinus  closinf^  by  j^ranu- 
lation.     Tlic  wound  had  been  dressed  surj-ioaily  every  third  day. 

'I'he  followin,!;-  eases  of  infoction  by  streptococcus  pyogenes  have  been 
treated,  with  the  results  recorded  : 

H.,  aged  57  years.  ^Malignant  endocarditis.  Two  months  before  admission 
to  hospital  felt  tired  and  weak.  Diarrhea  for  several  days.  Great  prostration; 
loss  of  appetite.  Three  weeks  later  lumbago.  Shortly  afterward  patient  had  a 
chill  with  a  temperature  of  103..^).  From  that  time  chills  and  fever  occurred 
daily.  Physical  examination  sliowed  mitral  and  aortic  systolic  murmurs  and 
many  petechia;  over  the  body.  A  streptococcus  was  found  in  the  blood  on 
November  3,  from  which  a  vaccine  was  made.  Inoculation  of  this  vaccine  was 
made  at  intervals  of  three  and  four  days,  beginning  with  1,000,000  sterile  organ- 
isms and  increasing  gradually  to  25,000,000.  The  evening  temperature,  which 
occasionally  reached  as  high  "as  103  and  averaged  102,  began  to  fall  after  the 
fifth  inoculation,  reaching  first  101,  then  averaging  100,  and  occasionally  remain- 


flay  o^i^^^  uiiixiAfkifiitaitft 


Lirtricnfirmj0  n  ftis  U  tftt  it 


Case  M.  S. 


ing  normal  with  rises  to  101.  On  December  8,  the  temperature  became  normal, 
remaining  so  for  four  days,  although  the  patient's  general  condition  was  not 
materially  improved,  the  diarrhea  remaining  very  troublesome.  On  December  13, 
the  patient  died  suddenly,  from  a  condition  diagnosed  as  "internal  hemorrhage," 
the  temperature  rising  to  103  just  before  death. 

M.  S.,  Malignant  Endocarditis;  Cerebral  Embolus.  Rheumatism  four  years 
ao-o.  Nine  days  before  admission  to  the  Roosevelt  Hospital  patient  had  cough 
and  oppression  in  praecordium  following  a  cold;  became  weak  and  had  chills; 
has  had  a  slight  dyspnea,  severe  headaches  and  marked  prostration.  Vomited. 
Had  felt  feverish  and  unwell  for  one  month  before  admission  to  hospital. 
Occasional  sweats  and  cardiac  palpitation. 

Physical  examination  shows  flattening  of  left  side  of  face.  Head  and  tongue 
turn  and  deviate  toward  right.  Presystolic  and  systolic  murmurs  at  the  apex  of 
the  heart.  Left  arm  and  leg  paralyzed.  Petechial  spots  in  skin.  Hyperesthesia. 
For  a  while  the  patient's  general  condition  improved.  Optic  neuritis  marked 
on   admission,   December    1,   continued   throughout.     There  were  ninnerous   crops 


24 

of  petechial  spots.  A  number  of  blood  cultures  gave  negative  results.  Leucocytes, 
at  first  26/200.  fell  in  one  week  to  10,000.  From  Dec.  1  to  Dec.  28,  1907,  the 
daily  temperature  ranged  from  102.4  to  normal  with  great  regularity.  On 
December  30,  2,000,000  of  a  streptococcus  vaccine  prepared  from  another  some- 
what similar  case  were  injected.  A  slight  fall  in  the  temperature  resulted  during 
the  next  two  days,  followed  by  a  rise  to  the  previous  temperature  after  a  second 
inoculation,  on  January  2,  of  4.000.000  sterile  streptococci.  Following  an  inocu- 
lation, on  January  6,  of  6,000,000,  the  temperature  was  lower  for  a  few  (Jays, 
but  after  an  inoculation,  on  January  10,  of  10,000,000,  the  temperature  rose  to 
103  for  two  days,  then  fell  to  normal  on  the  fourth  day  after  inoculation, 
remaining  about  normal  for  three  days  following  an  inoculation  of  5,000,000. 
After  another  inoculation  of  5.000,000,  four  days  later,  the  temperature  rose  to 
104.  at  wliich  time  symptoms  of  a  fresh  cerebral  embolus  appeared,  and  from 
this  time  the  temperature  remained  quite  uniformly  high.  No  more  inocula- 
tions were  given.  After  having  been  in  a  condition  of  coma  for  a  week,  the 
patient  died  February  5.     No  autopsy  was  obtained. 


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Case  A.  II. 


The  following  case  oi-  streptococcus  infection  of  Dr.  A.  J.  Bristow 

and  Dr.  W.  H.  Pratt  was  seen  with  Dr.  X.  B.  Potter : 

A.  H.  Female,  aged  58  years.  Dr.  A.  J.  Bristow  and  Dr.  W.  H.  Pratt, 
Brooklyn.  February  25,  rigors,  fever,  general  muscular  pains,  intense  headache. 
March  1,  well.  March  4,  repetition  of  previous  attack  with  more  severe  headache. 
March  10,  well.  March  11,  tenderness  over  left  side  of  neck.  Fever,  temperature 
100  to  103.  March  17,  moderately  circumscribed  swelling  of  left  submaxillary 
triangle.  No  fluctuation.  Leucocytes  15,000,  polyiuiclears  78  per  cent.  March 
18,  swelling  less  painful,  less  tender.  Leucocytes  12.000,  polynuclears  78  per  cent. 
March  20,  swelling  more  diflFuse,  more  painful.  ^larch  21.  leucocytes  10,000, 
polynuclears  75  per  cent.  March  23,  swelling  had  extended  to  clavicles,  more 
tender.  Leucocytes  11,000,  polynuclears  83  per  cent.  A  long  incision  parallel 
with  anterior  border  of  sternocleidomastoid,  tissues  much  thickened  and  infil- 
trated. Deep  in  submaxillary  fossa  small  abscess  cavity  opened,  pus  evacuated, 
drainage.  Culture:  Htrept.  longns  and  a  bacillus.  March  24.  edge  of  wounds 
glazed.  Very  little  discharge.  Urine  normal.  March  20,  intermittent  pulse. 
March  27,  severe  pain  in  left  leg.  Some  tenderness  over  internal  condyle.  March 
28.  infiltration  of  neck  slowly  disappearing.  March  30,  two  slight  convulsions 
limited  to  face  and  upper  extremities,  eyes  to  right.     Face  livid.     Blowing  niur- 


25 

mur  over  mitral.  Marcli  31,  pain  in  ri<T;ht  knoo  and  left  forefinfjor  and  wrist. 
April  1,  seen  by  Dr.  Potter.  Opsonic  inde.K  to  streptococcus  A.  R.  (Dr.  Krum- 
wiede),  0.74.  April  2,  blood  culture  (Dr.  X.  E.  Ditman)  sterile.  Inoculated 
with  25.000,000  streptococci  A.  R.  (Vaccine  prepared  by  Dr.  Kruniwiede. )  April 
4.  wound  diminished  in  size  one-half.  Joints  have  improved.  Inoculated  with 
50,000,000.  April  11,  wound  practically  healed.  Convalescence  uninterrupted. 
The  striking  features  of  tliis  case  are  the  improvement  of  the  wound  and  joint 
SAinptoms  within  forty-eight  hours  after  the  first  dose,  and  the  sudden  drop  in 
the  temperature  and  Vapid  improvement  following  within  forty-eight  hours  after 
the  second  ino<'ulation  and  closelj-  corresponding  to  the  rise  in  the  opsonic  index, 
as   plainly  shown  in   the  chart. 

A  severe  case  of  puerperal  septicemia  resulting  from  uterine  infection 
with  streptococcus  p3^ogenes,  which  was  running  a  high  septic  tempera- 
ture, was  inoculated  with  50,000,000  of  sterilized  streptococci.  Death 
occurred  one  week  later,  no  improvement  having  been  noted  at  any  time. 

The  following  cases  of  gonorrheal  infection  have  been  treated : 

P.  Gonorrheal  Arthritis.  Three  years  previous  to  the  present  period  of  treat- 
ment, he  had  a  gonorrheal  urethritis  which  was  never  cured.  Two  years  ago 
he  developed  an  arthritis  in  the  left  knee,  as  well  as  in  the  right  knee  and  both 
ankles  in  milder  form.  Massage,  baking,  and  antigonococcic  serum  had  been 
tried  without  effect.  Function  had  become  more  and  more  impaired  until  the 
patient  was  confined  to  bed.  Tlie  knee  joint  had  been  opened  and  treated  sur- 
gically. The  joint  healed  in  a  stiffened  condition  and  was  much  swollen,  due 
to  an  inflammatory  infiltration  of  the  synovial  membranes.  An  abscess  developed 
imder  the  tendo  achillis  which  was  incised  and  drained,  but  refused  to  heal. 
The  evening  temperature  reached  100  daily,  and  the  knee  joint  was  much  swollen 
and  exceedingly  painful,  no  active  motion  being  possible.  On  October  27,  an 
inoculation  of  a  vaccine  containing  2. .5  million  gonococci  was  given.  Two  days 
later  the  temperature  became  normal,  and  the  pain  in  the  knee  was  less.  Five 
days  later,  5,000,000  gonococci  were  given,  followed  at  increasing  intervals  by 
doses  of  10,  15,  25,  50,  100  and  100  million.  Shortly  after  treatment  was 
begun,  the  sinus  healed.  Pain  disappeared  from  the  knee  in  three  weeks  and 
active  motion  was  begun.  Congestion  was  caused  at  intervals  by  hot  local 
applications  followed  by  centripetal  massage  to  renew  the  serum  supply  in  the 
joint.  On  January  1,  the  joint  had  returned  to  normal  size  and  normal  function 
had  been  regained. 

M.  W. — Gonorrheal  Arthritis.  Admitted  to  the  Roosevelt  Hospital,  November 
9,  1907.  Two  days  before  admission,  pain,  first  in  back  and  legs,  then  in  arms, 
neck  and  head.  Pains  then  became  general  and  patient  was  sore  to  the  touch. 
She  had  a  shaking  chill  followed  by  a  marked  sweat,  and  since  has  felt  weak 
and  feverish.  There  was  general  tenderness  of  the  lower  extremities  and  four 
days  after  admission  fluid  was  detected  in  the  right  knee.  The  temperature 
ranged  from  98.6  to  102.  Aspirin  was  given  without  effect.  Leucocytes,  14,000. 
On  November  10,  the  right  knee  became  worse,  and  the  patient  looked  much 
sicker.  For  about  forty  days  the  temperature  reached  100  or  101  daily. 
November  29,  right  knee  swollen  and  painful,  no  redness  or  heat.  November  23, 
gonococci  were  isolated  from  aspirated  joint  fluid.  At  this  time  an  antigono- 
coccus  serum  was  injected.  December  2,  slightly  less  pain  in  joint.  December 
17,  tuberculin  reaction  negative.  Bier's  treatment  commenced  December  18;  no 
improvement.  January  29,  the  right  knee  was  still  considerably  swollen  and 
excruciatingly  painful.  Motion  of  any  kind  was  impossible  and  sleep  was  almost 
impossible  on  account  of  pain.  Two  and  a  half  million  of  a  polj-valent  gono- 
coccus  vaccine  was  given.  On  February  3,  5,000,000  were  given;  on  February  7, 
10.000,000;  on  February  11.  20,000,000,  on  February  15,  50,000,000,  on  February 


26 

21,  100.000.000.  and  on  iMaicli  7.  100,000.000.  After  the  second  injection  pain 
diminislied  sutlieicntly  to  allow  restful  sleep,  and  after  each  successive  injection 
the  pain  became  less  until  after  tlie  fourth  there  was  no  pain  on  palpation. 
The  circumference  of  the  knee  at  upper,  middle  and  lower  parts  of  patella 
diminished  as  follows,   from   February   1    to  ]\Iarch   8: 

February    1 131^  inches  13%  inches  13i^  inches 

:M{irch    8 l-iy^  inches  13       inches       ^     I214  inches 

In  reviewing  the  preceding  series  of  cases  the  deduction  seems  reason- 
able that  the  etficiency  of  inoculation  by  bacterial  vaccines  depends  upon 
the  intensity  of  the  infection  and,  more  especially,  upon  whether  the 
infection  is  local  or  systemic.  Virulent  infections  are  benefited  with 
difficulty,  and  while  unifonn  benefit  seems  to  follow  the  administration 
of  vaccines  in  local  conditions  of  infection,  little  or  no  benefit  seems  to 
result  if  the  infection  is  systemic,  under  conditions  where  the  organisms 
exist  in  numbers  free  in  the  blood  stream. 

During  recent  years  the  possibility  of  obtaining  l^eneficial  therapeutic 
results  in  tubercular  infections  by  the  inoculation  of  tuberculin  has  re- 
ceived considerable  attention.  The  forms  of  tuberculin  used  are  as  fol- 
lows :  Koch's  old  tuberculin  is  made  by  evaporating  by  heat  liquid  cul- 
tures to  one-tenth  of  their  original  volume  and  filtering  off  the  bacilli 
through  a  porcelain  filter.  His  two  more  recent  tuberculins,  T.E.  and 
B.E.,  represent  attempts  at  producing  antitoxic  and  antibacterial  im- 
munity and  are  suspensions  of  crushed  tubercle  bacilli  in  glycerin  and 
water.  B.F.  is  merely  a  filtrate  of  human  cultures  of  tubercle  bacilli  of 
recorded  virulence.  These  tuberculins  are  all  "vaccines."  The  two  chief 
methods  of  tuberculin  inoculation  are  that  of  "Wright  and  the  '•'clin- 
ical" method  or  methods  of  Denys.  Wright  uses  constant  sized  small 
doses,  about  1/100  mgm.  of  T.R.  and  regulates  the  intervals  by  tlie 
opsonic  index. 

In  the  "clinical"  method  the  dosage  at  first  is  very  small,  1/10000 
mgm.,  and  is  increased  gradually  at  intervals  of  three  or  four  days  by 
sufficiently  small  doses  to  avoid  any  "reaction,"  general  or  local. 

Some  express  preference  for  B.E.  tuberculin,  which  contains  the 
toxins  and  endotoxins  to  be  found  in  the  bodies  of  the  crushed  germs, 
entire  and  unaltered  by  heat  or  chemicals,  and  produces  antibacterial  and 
antitoxic  immunity.  With  this  tuberculin,  habituation  takes  place  with 
much  more  difficulty  than  with  B.F.,  and  occasionally  unexpected  and 
sometimes  violent  reactions  occur,  even  if  the  utmost  caution  in  increasing 
the  dose  is  exercised.  For  this  reason  B.F.  is  considered  bv  many  prefer- 
able, as  it  is  easier  to  control  its  effects.  Up  to  the  present  time  the 
method  of  Wright  has  been  largely  confined  to  local  tubercular  processes. 

For  tlie  treatment  of  pulmonary  tuberculosis  the  "clinical""  method, 
or  iiictli(i(l  (if  I'cnys,  has  been  largely  used. 


27 

The  following  series  of  cases  illustrate  this  method  of  tubercnlin 
therapy,  the  temperature  curve  constituting  one  of  the  most  important 
guides  to  systemic  effect,  the  opsonic  index  not  being  employed : 

W.    H..  aged   34  years.    Chronic   Pulmonary   Tuberculosis.     Had    an    attack   of 
inllucnza  six  months  before  apply iny-  for  treatment.     Since  then  he  has  coughed 


month    \    (U^.           iJfMitr 

0'3^A/r.              H«u««<JtU^ 

Day  of     1    . 

102  -* !5 

-*: ■«= 

^    lof- 5 

g         

__2, ^ 

-—^  —  —  ~  —  —  —  ^  —  —  —  i^  —  — 
-  — ^ 3 IB 

-•EEEEEEEEiiiEil 

Q7-]    1        1        1    1    [                              f 

Case  W.  II. 


Month       %J^.                       \^*^ 
Day  of                                          ^ 

103° -p i 

102" * 

H     Mo  4  H        p 

T«ir.     .                 ^K^. 

^   100"-^ ' 

E 

(2                                                           : 
99c 

kU — ^ — i- 

Case  W.  II. 


continually,  with  mucoid  expectoration  and  pain  in  the  right  chest.  Some  fever 
and  occasional  night  sweats.  One  hemoptysis.  Over  the  apex  of  the  right  lung 
the  percussion  note  was  dull,  breathing  bronchovesicular,  with  a  few  subcrepitant 
rales   at  both  apices.     His  temperature  rose  every   afternoon  to   100,   falling  to 


28 


normal  in  tlie  morning.  Injections  of  tuberculin  (bouillon  filtrate,  made  at  tbe 
Trudeau  Sanatorium  by  Dr.  Baldwin,  and  kindly  furnished  by  him)  were  begun 
August  9.  At  first  .000,000,1  gm.  was  given;  then  the  doses  were  increased  in 
size  progressively,  one  every  four  days,  the  progression  being   .000.000.2  gm.  up 

Moath     lACC* 


Day  of   Im  ^  16  It  tus  14  iS  ifc  ii  IS  1^  Itv  t^^^utf^fct1tgtf  toSi  /  1  ^  4  ^ 


ft^ 


Case  T.  O'M. 


to  .000.001  gm.,  then  by  .000,002  gm.  up  to  .000,01  gm.,  then  by  .000,02  gm. 
up  to  .000.1  gm.,  then  by  .000.2  gm.  up  to  .001  gm.,  which  point  was  reached 
December  20,  without  any  reactions  having  been  experienced.     By  November   9, 


Month       \v\.— 

TA. 

Dayof      Xa^  k  1  14   »«    it  »J /4 /^ /# /7 /f 

infs° ' 

^    iof:o-e Si — j5 — =±S- 

L    .1  y  uj 

—  s ^  —  ^          aos 

-^-^S: — :5^ 5ai' 

_5 s :s «^-« 

^   100^—^ \. —  ^^ :->- 

E             

s 1 i : •-»—.• 

. 

^ 

Case  T.  OM. 

all  pathological  physical  signs,  and  cough  and  pain  had  disajjpcarcd.  and  by 
December  1:5,  the  temperature  had  fallen  to  normal,  to  remain  there.  The  gain 
in  weight  had  been  eight  pounds,  and  the  patienfs  general  condition  was  excel- 
lent.    Cod  liver  oil  with  malt  was  given  througliout. 


29 

T.  O'M.,  aged  19  years.  Clnonie  Pulmonary  Tuberculosis.  For  eiglit  months 
before  presentation  for  tieatment,  jjutient  had  eough,  indigestion,  pain  in  left 
chest.  Has  lost  twenty  pounds.  Has  had  mucopurulent  expectoration,  but  no 
hemoptysis.     Some  dyspnea  and  fever.     Two  (piarts  of  clear  fluid  were  withdrawn 


iiK%ti^l<^i^v  \  1  i  4  f  » lir  <  f  H  Alt  m.if  *n  /rif  w^  u;tu 


Case  T.  O'M. 

from  the  left  chest.  There  was  slight  dullness  over  the  right  apex  and  the 
whole  left  chest  with  subcrepitant  rales  over  the  same  area.  Before  the  begin- 
ning  of   treatment   the   temperature    frequently  rose  to  lOO.G.    After  the  inocula- 


Month 
Day  of 


Lg  tfcti  Uti^tot  ri  >  4  f  fc  7  y  n  (f,[|  a(S,iA>r/t,l7 


103' 


102' 


101 


100 


99' 


98 


97 


hM. 


Vft*" 


:s» 


wmmm 


i?=2 


i^nttui^ 


^iCI 


5^=G52ZgSe 


V 


Case  T.  OM. 


tions  of  tuberculin  (B.F.)  were  begun,  Dec.  IG,  1907,  the  temperature  did  not 
rise  above  99,  and  after  January  6,  remained  at  normal  or  below.  The  cough 
diminished  markedly,  great  improvement  in  the  patient's  general  condition 
resulted,  and  by   February   17   he  had  gained   ten  and  one-half  pounds.     At    this 


30 

lime  tlio  same  ])ortions  of  the  luiijis  were  still  affected,  but  the  rales  had  become 
drier  and  less  iiunioroiis.  Cod  liver  oil  with  malt  was  administered  throiij^hout 
tlie  treatment. 

A.  S..  aged  33  years.  Chronic  Pulmonary  Tuberculosis.  For  three  years  this 
\\oman  had  a  cough;  at  the  end  of  the  first  year  had  hemoptysis.  Cough  got 
steadily  worse;  fever  developed,  but  no  night  sweats.  Dyspnea  developed  gradu- 
ally, and  shortly  before  presentation  for  treatment  she  felt  pain  in  the  right 
chest.  For  one  year  she  has  had  diarihea.  and  during  the  past  two  years  her 
weight  has  fallen  from  130  to  1 11  pounds.  Tubercle  bacilli  Avere  found  in  the 
sputimi. 

A  course  of  tuberculin  inoculations  (B.F. )  was  begun  on  Xov.  26.  1907,  and 
tlie  dosage  increased  regularly,  until  on  January  27  the  dosage  was  0.000,1  grams. 
Tlie  temperature,  which  at  first  rose  to  99  daily,  remained  below  normal  after 
•January  9.  Tliere  was  a  marked  improvement  in  appetite  and  general  appear- 
ance, and  by  Januaiy  27,  she  had  gained  ten  pounds.  The  cough  was  much 
improved,  but  tubercle  bacilli  were  still  present  in  the  sputum.  At  first  there 
was  slight  dullness  over  the  upper  lobe  of  the  right  lung,  with  many  subcrepitant 
rales,  almost  to  the  base.  On  January  27,  the  dullness  had  markedly  diminished 
and  the  subcrepitant  rales  Avere  confined  to  the  right  apex.  Cod  liver  oil  with 
u.alt  was  given  throughout   the  treatment. 


Month 
Day  of 


(^.  "KfN-. 


_  tU        K>       TUr. iJiAsr.^ 


Case  M.  L. 


Mj.  L.,  aged  48  years.  Chionic  Pulmonary  Tuberculosis.  For  two  years  this 
man  had  a  cough  with  pain  in  the  chest,  mucopurulent  expectoration,  loss 
of  weight,  weakness,  slight  fever — at  times  reaching  to  100.  Physical  exami- 
nation showed  slight  dullness  at  tha  right  apex  of  the  lung  with  many  sub- 
crepitant rales  at  both  apices.  Tuberculin  (B.F.)  inoculations  were  begun  Aug. 
19.  1907,  by  the  usual  method,  and  were  being  continued  ilarch  13,  1908.  when 
the  dosage  had  reached  0.001  grams.  The  temperature  has  gradually  returned 
to  normal,  the  cough  has-  ceased,  and  there  has  been  a  gain  of  ten  pounds  in 
weight  with  most  marked  improvement  in  the  patient's  general  apix-arance  and 
condition.  The  physical  signs  have  become  normal  with  tlu^  exception  of  an 
occasional  dry  click  at  one  side.  Cod  liver  oil  with  malt  was  administered 
tlnoughout  the  treatment. 

S.  McL.,  aged  20  years.  Cluonic  Pulmonary  Tuberculosis.  Ten  weeks  before 
ol)servation  on  tliis  patient  commenced,  she  caught  cold  and  developed  a  con- 
tinued cough  with  ])ain  in  the  side,  blood  streaked  sputum,  which  was  afterward 


31 


found  to  contain  tnberclp  bacilli,  fever,  and  great  loss  of  weight.  Well  marked 
signs  of  dullness  on  percussion  were  found  at  the  left  apex  with  bronchial  voici 
and  numerous  subcrepitant  rales.  She  was  confined  to  bed  in  St.  Ijuke's  Hospital 
for  three  weeks,  witli  much   improvement,  and  on  Dec.  2,   1007,  received  the  first 


Month 
bay  of 


I 


u. _^  h^ 


103' 


102' 


101' 


100 


99" 


98 
97' 


mm 


a=:te 


iiiiliii 


f? 


i 


5r' 


?5? 


>a 


•^^r 


Case  S.  McL. 

of  a  series  of  tuberculin  (B.F. )  inoculations,  given  by  the  usual  clinic<il  method 
at  four-day  intervals  in  progressive  doses.  The  fever,  which  at  first  reached 
as  high  as  99,  became  normal  from  December  31,  the  cough  stopping  at  about 


Month       V*^                                                                          ^d^. 

Day  of      /JnTl  ,S  1^  |6tltl  Utt  HK'tiV  l^ijUtt    S  4  i"  *  7  8 

103° 

102"__:zte___ ^___,_^___^ 

^ ^ B_ ^_^ o 

I      101 ^:— i ^ ^-^ Tl- 

g             n — _^ L^ ^  —  ^ ^ 

S.    100° §  —  ^ ' — ^ -•— fe -t^ 

B            . . . . 

99' \ 

97\                  ^                                       =^ 

Case  S.  McL. 


the  same  time.  By  February  7,  the  physical  signs  had  become  normal  and  the 
patient  had  gained  twenty  pounds  since  first  presenting  herself  at  the  hospital. 
General  condition  had  become  excellent.  Cod  liver  oil  with  malt  was  adminis- 
tered throughout. 


"M.  C.  aged  43  years.  Chronic  Pulnionarv  Tuberculosis.  Had  a  cough  for 
one  year  before  presenting  lierself  for  treatment.  Profuse  expectoration,  some- 
times bloody;  no  profuse  hemorrhages;  night  sweats.  Dyspnea  and  occasional 
pain  in  chest.  Appetite  very  poor.  Used  to  weigh  135  pounds;  weighed  lOlVj 
jiounds  when  treatment  was  begun.  At  this  time  there  was  a  general  tubercular 
iiitiltralion  of  the  entire  left  lung  with  uniform  dullness  on  percussion  and 
numerous  moist  snbcrepitant  rales.  A  few  subcrepitant  rales  were  heard  at 
tiie  right  apex.  The  pulse  rate  was  uniformly  rapid  (128)  and  the  daily  range 
of  temperature  was  100.0  to  07.  The  general  condition  was  very  poor.  Inocu- 
lations of  tuberculin  (B.F.)  were  began  Aug.  16.  1907,  by  the  clinical  method. 
iK^sinning  with  0.000.000,1  gm..  and  were  continued  uniformly  until  January  G, 
at  which  time  the  dosage  had  reached  0.008  gm.  Until  December  8.  there  was 
little  change  in  the  patient's  condition,  but  from  that  time  on  the  evening 
temperature  began  to  fall,  and  from  December  23  on.  the  evening  temperature 
was  not  over  98.4.  The  loss  of  weight  ceased  after  she  had  fallen  to  98  pounds 
and  the  weight  .sub.sequently  rose  to  100  pounds.  The  most  marked'  change  was 
in  the  patient's  general  condition,  which  had  improved  markedly,  and  in  the 
improvement  of  the  cough.  The  abundance  of  moist  rales  were  replaced  by  a 
few  dry  rales.  On  February  1,  the  patient  slipped  on  the  ice.  fell  and  suffered 
a  severe  concussion,  being  confined  to  bed  for  six  weeks,  during  which  time  her 
physician,  for  some  reason,  submitted  her  to  frequent  and  severe  purging  and 
a  iow  diet.  While,  following  this,  there  was  no  rise  of  temperature,  her  weight 
was  reduced  to  96  pounds,  and  the  pulmonary  rales  became  less  dry.  The  lesion 
at  the  right  apex,  which  had  disappeared,  still  remained  absent.  During  her 
entire  treatment  cod  liver  oil  with  malt  was  given. 

By  this  method  improved  results  of  treatment  of  pidmonary  tuber- 
culosis have  been  shown  by  Trudeau,  who  finds  that  18  to  25  per  cent, 
or  more  treated  than  of  untreated  cases  discharged  from  the  sanatorium 
during  fifteen  years  were  living  at  the  time  of  inquiry. 

The  author's  cases  are  too  few  in  number  to  compute  any  percentage 
improvement,  but  the  rapid  improvement  in  the  patients'  condition  and 
the  uniformity  of  benefit  can  not  be  considered  anything  else  but  encour- 
aging in  adopting  this  method  of  therapy. 

AVright  has  published  results  of  treatment  of  a  number  of  cases  of 
localized  tuberculous  infection  by  means  of  inoculation  of  small  doses  of 
Koch's  new  tuberculin.  The  cases  reported  thus  far  include  tuberculous 
peritonitis,  cystitis,  glands  and  sinuses  following  an  operation  for  their 
removal,  tuberculosis  of  tlie  subcutaneous  tissues,  lupus,  etc.  He  has  met 
with  astonishingly  good  results  in  a  very  large  proportion  of  cases,  and 
his  reports  have  done  much  to  reinstate  Koch's  new  tuberculin  as  a  thera- 
peutic remedy.  The  effect  of  tuljcrculin  inoculations  on  tlie  opsonic 
index  in  two  of  our  cases  is  exhibited  in  the  following  charts,  showing 
the  ^^riation  in  the  opsonic  indices  during  treatment. 

IXFLUEXCK  OF   TIEREDITAi; V   TKXDEXCV   OX   TUBERCULO-OPSOXIC   IXDEX. 

Tbe  (juestion  wlietlier  low  indices  are  the  cause  or  the  effect  of  the 
infection  is  one  of  considerable  interest.  The  tul)ercido-o])sonic  indices 
of  a  numl)er  of  appai-ently  liealthy  children  of  tuberculous  parents  are  as 
follows: 


33 

Table  of  Children  of  Tuberculous  I'arents. 

April  lil. 

M.    D (i.s.-) 

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M.    J,,    aged    35.     Tuberculosis    of    hip    joint    treated    with    small    doses    of    T.U. 
Result  :      marked    improTement. 


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Case   of   tuberculous   ankle.      NO    improvement    clinicn  lly. 


Several  low  indices  occur  in  this  series.  In  a  nnniber  of  picked  cases 
of  "sanitarium  cures"  of  phthisis  in  its  early  stages  Bulloch  constantly 
found  low  indices.  Here,  again,  it  would  be  important  to  know  Avhether 
such  cases  relapsed,  as  so  frequently  happens.  Bulloch  believes  the  low 
index  probably  due  to  some  hereditary  or  acquired  imperfection  in  the 


34 

mechanism  by  whicli  antibacterial  substances  are  mamvfactiired.  In  this 
connection  it  is  interesting  to  note  that  Simon  has  recently  found  the 
opsonic  content  of  serum  to  be  diminished  in  some  cases  by  starvation. 

PRACTICAL  VALUE  OF  OPSONIC  IXDEX. 

In  reviewing  tlie  work  wliieh  lias  already  iK'on  accomplished  on 
opsonins-*^  the  question  of  wliat  practical  use  they  may  have  is  one  of 
greatest  interest. 

Tlie  great  difficulty  will  arise  from  the  fact,  probably,  that  changes 
more  or  less  profound  are  indicated  at  times  only  by  slight  variations  of 
the  indices,  and  that  the  variation  is  frequently  not  far  beyond  the  limits 
of  error  in  a  method  where  opportunities  for  small  errors  in  technic  are 
numerous. 

Some  aid  in  diagnosis  may  be  had  by  determination  of  single  indices; 
but  before  this  can  be  considered  a  reliable  method  considerable  work 
must  be  done  to  determine  the  degree  of  specificity  of  opsonins.  AVright 
and  Eeid's  work  indicates  that  some  diagnostic  aid  may  be  obtained  by 
comparison  of  serum  and  exudates,  and  by  the  methods  of  heated  and 
unheated  serum. 

A  wider  field  of  application  will  probably  be  in  the  control  of  inocu- 
lation of  vaccines  to  determine  the  optimum  dosage  and  regulation  of 
frequency  of  the  same,  but  this  will  be  somewhat  limited  by  the  clinical 
difficulties  of  the  method. 

A  field  as  yet  but  little  explored,  in  which  it  may  prove  of  great  use, 
is  in  the  determination  of  susceptibility  to  infection,  especially  in  the 
inherited  susceptibility  to  tuberculosis. 

Perhaps  its  greatest  usefulness  will  be  found  in  the  laboratory,  wdiere 
it  may  prove  to  be  a  convenient  and  rapid  method  of  testing  the  virulence 
of  organisms  and  the  strength  of  sera  which  are  dependent  on  opsonins 
for  their  beneficial  effects. 


48.  For  additional  literatm-e  the  reader  is  referred  to:  Wright:  •"Treatment 
of  Fnrunculosis.  etc..  by  Inoculation  of  Staphylococcus  Vaccine."  Lancet.  March  2U. 
1002;  also  "Treatment  of  Acne  Furunculosis,"'  Brit.  Med.  Jour.,  May  7.  1004;  "Inocu- 
lation Treatment  of  Tuberculosis,"  Clinical  .Journal,  May  7.  1904 ;  "Inoculation 
Treatment  of  Tuberculosis,"  Clinical  Journal.  Nov.  0,  1904 :  "The  Serum  Reactions 
vol.  Ixsvii,  p.  104 ;  "Therapeutic  Inoculation  of  Bacterial  Vaccines,"  Brit.  Med. 
Jour.,  May  9,  190;^  :  "Certain  New  Methods  of  Blood  Examination  with  Some  Indica- 
tions of  Their  Clinical  Importance,"  Lancet,  Jan.  23,  1904,  p.  215  ;  "A  Short  Treatise 
on  Antityphoid  Inoculation,"  London,  1004;  "Measurements  of  liacterial  Power  of 
Blood."  I'roc.  Koyal  Soc,  1002,  August,  vol.  Ixxi,  p.  54  ;  "I*i-ocedures  for  Testing 
and  Judging  by  Naked  Eye  of  Agglutinating  and  Bacteriolytic  Effects  Exerted  by 
Sera  (Spontaneous  Agglutination),"  Lancet,  1003.  July  25.  p.  214;  Lancet.  1004. 
Aug.  (!,  p.  411;  Wright  and  IJeid  :  Proe.  Boyal  Soc,  lOOfi.  vol.  Ixxvii.  p.  104: 
Wright  and  Douglas:  "Tuberculosis,"  Lancet,  Oct.  22,  1004,  p.  113S  ;  I'.u'.loch  and 
Atkin  :  "Experiments  on  the  Nature  of  the  Opsonic  .\ction  of  the  Blood  Serum." 
Proc.  IJoyal  Soc,  1005,  vol.  Ixxiv,  p.  379;  Smith,  R.  Greig :  "Agglutination  and 
Lf-ucocytosis,"  Proc.  Linn.  Soc.  New  South  Wales,  1005,  part  2,  July  2t!  ;  Cheyene  : 
"Wright's  Method  of  Treating  Tuberculosis,"  Lancet.  Jan.  13,  100(>,  p.  78  ;  I'ark  and 
Williams:  Jonr.  Exp.  Med..  1005,  vol.  vii,  p.  403;  Craw,  J.  A.:  "Mechanism  of 
Agglutination."  Jour,  of  Hyg.,  January,  1905,  vol.  i.  p.  12(5;  Houston:  "Dermatologii 
'I'herapeutics."  Brit.  Med.  Jour.,  1005,  vol.  i,  p.  S54  ( .\  Reply):  Pearce  .uid  Winne- 
Amer.  Jour.  Med.  Sci.,  October,  1004. 


35 

111  sj)ilc  of  tile  fad.  Iit)\\  <'\i'i-.  llial  llic  niftlniil  for  llic  (lclci-|ii  iiial  ioii 
of  till'  opsonic  iinlcx  lia>  iim  |ir()\cii  niii'  of  <j:vcii[  cxacliicss  in  any  oni' 
case,  by  tlic  fk'trnninalioii  of  a  lai-iir  minilicr  of  imiiccs  in  cases  wiiicli 
have  uudtTuoiic  iiiociilal  imis  liy  liactcrial  \accincs.  it  has  hccii  [Missihlc 
to  dotcniiiiit'  Ihc  opliiiiiini  size  of  ihc  doses  and  ihi'  iiincidalioii  inlei-\als 
for  all  the  various  ty|ies  n\'  oriiaiiisnis  eiii|ilo\  (mL  Ai  ihe  |)reseiii  lime, 
iherefore.  our  kno\\ledi;e  ol'  these  iiiatlei-s  is  such  ihal  h\-  I'eKiii;^  on  iioili- 
ino-  more  than  the  clinical  symptoms  haeterial  vaccines  may  he  a(h]iiii- 
istored  in  rsafety  and  with  benefit. 

Their  use  in  systemic  infections  will  |)i'ohal)ly  rarely,  if  ever,  result 
in  very  satisfactory  results,  hut  for  their  use  in  localized  infections,  even 
in  such  vascular  viscera  as  the  liinos,  thei-e  is  promise  of  results  in  ad- 
vance of  many  therapeutic  methods  now  in  use. 

in  coni-lusion.  we  desire  to  thank  I)rs.  Ewin.u-,  Wadsworth,  Theohahl 
Smith,  lialdwin.  Twitchell.  Dunham  and  Torrey  for  hactei'iolooic  ma- 
terial supplied.  We  also  desire  to  acknowledL;-e  ycyy  capable  assistance  on 
the  pari  of  Dr.  Millicent  Hopkins. 


l.-iiiriiitrd   ill    1,(1,1    from    flic  .loiiniul   of    the   Aiiurican    Mcilknl    As.iocintioii,    Xi 
."/.    won.    Vol.  alril,  pp.   n22-2o,    and   Decniihrr    I.    W06, 
Vol.  a-liii,  pp.   nOS-lHOO. 


AiiKiifuii    Midiriil    A.-i.^iji  iiitioii    Prr.s-.s.    /".,'    iHiirboiii    Arc,    CJiivdfjo. 


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